CD4-independent HIV envelope proteins as vaccines and therapeutics

ABSTRACT

The invention relates to novel CD4-independent HIV Envelope proteins and uses therefor.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/317,556 filed May 24, 1999.

STATEMENT REGARDING FEDERAL SPONSORSHIP

[0002] This invention was supported in part by funds from the U.S.Government (National Institutes of Health Grant No. AI44308 and GrantNo. AI40880) and the U.S. Government may therefore have certain rightsin the invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to CD4-independent variants of HIV,their proteins, and uses therefor.

[0004] HIV entry is known to require an interaction of the viralenvelope glycoprotein (Env) with CD4 and cellular chemokine receptors.HIV Env protein is produced as a precursor (gp160) that is subsequentlycleaved into two parts, gp120 which binds CD4 and chemokine receptors,and gp41 which is anchored in the viral membrane and mediates membranefusion. Differential use of chemokine receptors by HIV and SIV haslargely explained differences in tropism among different isolates(Berger, 1997, AIDS 11:S3-S16; Hoffman and Doms, 1998, AIDS 12:S17-S26).While a number of chemokine receptors can be utilized by HIV or SIV(Deng et al., 1997, Nature 388:296-300; Choe et al., 1996, Cell 85,1135-1148; Rucker et al., 1997, J. Virol. 71:8999-9007; Edinger et al.,1997, Proc. Natl. Acad. Sci. USA 94:14742-14747; Liao et al., 1997, J.Exp. Med. 185:2015-2023; Farzan et al., 1997, J. Exp. Med. 186:405-411),CCR5 and CXCR4 appear to be the principal coreceptors for HIV-1 (Zhanget al., 1998, J Virol. 72:9337-9344; Zhang et al., 1998, J. Virol.72:9337-9344.). Isolates of HIV that first establish infection targetblood lymphocytes and macrophages using CCR5 (Alkhatib et al., 1996,Science 272:1955-1958; Deng et al., 1996, Nature 381:661-666; Dragic etal., 1996, Nature 381:667-673; Doranz et al., 1996, Cell 85:1149-1158),while viruses that are generally associated with progression to AIDS andcan infect T cell lines in vitro use CXCR4 (Choe et al., 1996, Cell85:1135-1148; Feng et al., 1996, Science 272:872-876; Connor et al.,1997, J. Exp. Med. 185:621-628).

[0005] Binding of Env to CD4 initiates poorly understood conformationalchanges enabling gp120 to bind to a chemokine receptor and leading tofusion of the viral and cellular membranes (Jones et al., 1998, J. BiolChem. 273:404-409; Moore et al., 1994, J. Virol. 68:469-484; Wyatt,1992, J. Virol. 66:6997-7004; Wu et al., 1996, Nature 384:179-183).Immunologic and mutagenesis approaches have indicated that these changesinvolve movement of V1/V2 and V3 hypervariable loops on gp120 (Moore, etal., 1994, J. Virol. 68:469-484; Wyatt et al., 1992, J. Virol.66:6997-7004; Wu et al.,1996, Nature 384:179-183), which play a criticalrole in the specificity of chemokine receptor utilization (Choe et al.,1996, Cell 85:1135-1148; Cocchi et al., 1996, Nature Med 2:1244-1247;Cho et al., 1998, J. Virol. 72:2509-2515; Speck et al., 1997, J. Virol.71:7136-7139; Ross et al., 1998, Proc. Natl. Acad. Sci. U.S.A.95:7682-7686; Hoffman et al., 1998, Proc. Natl. Acad. Sci. U.S.A.95:11360-11365). The recent crystallographic resolution of a gp120 corestructure bound to CD4 has revealed an intervening β sheet (the“bridging sheet”) between the inner and outer domains of gp120 that mayserve as an additional contact site for the chemokine receptor (Wyattand Sodroski, 1998, Science 280:1884-1888; Rizzuto et al., 1998, Science280:1949-1953).

[0006] Although CD4 is generally required for gp120 to associate with achemokine receptor, the identification of CD4-independent isolates ofHIV-1, HIV-2, and SIV has demonstrated that functional interactions withchemokine receptors can occur in the absence of CD4 interaction (Edingeret al., 1997, Proc. Natl. Acad. Sci. USA 94:14742-14747; Reeves andSchulz, 1996, J. Virol. 71:1453-1465; Endres et al., 1996, Cell87:745-756; Dumonceaux et al., 1998, J. Virol. 72:512-519). Thedeterminants for the CD4-independent phenotype have been mapped to theviral env gene, but the underlying mechanisms of this phenotype areunknown. It has been proposed that mutations in env may increase theexposure and/or the affinity of the chemokine receptor binding site ongp120, thus circumventing the need for CD4 (Endres et al., 1996, Cell87:745-756).

[0007] Biochemical assays have also shown that mutated or deglycosylatedrecombinant gp120 can bind directly to chemokine receptors, suggestingthat domains normally activated by CD4 can be artificially exposed(Hesselgesser et al., 1997, Curr. Biol. 7: 112-121; Martin et al., 1997,Science 278:1470-1473; Bandres et al., 1998, J. Virol. 72:2500-2504;Misse et al., 1998, J. Virol. 72:7280-7288). A greater understanding ofthe determinants responsible for CD4-independence should provideinsights into the Env domains that mediate and modulate interactions ofEnv with chemokine receptors and that ultimately govern viral entry.

[0008] To date, the ability of HIV-1 to escape the immune system hashindered development of efficacious vaccines to this important humanpathogen. Thus, there is a long-felt and unfilled need for thedevelopment of effective vaccines and therapeutic modalities for HIV-1infection in humans. The present invention meets those needs.

BRIEF SUMMARY OF THE INVENTION

[0009] The invention includes an isolated nucleic acid encoding aCD4-independent human immunodeficiency virus-1 (HIV-1) env, or a mutant,derivative, or fragment thereof. In one aspect, the isolated nucleicacid shares at least about 98% homology with the nucleic acid having thenucleotide sequence of SEQ ID NO:4.

[0010] In another aspect, the nucleic acid is selected from the groupconsisting of an HIV-1/IIIBx env, and an HIV-1/IIIBx 8x (8x) env.

[0011] In yet another aspect, the nucleic acid is an HIV-1/IIIBx 8x env.

[0012] The invention also includes an isolated nucleic acid encoding aCD4-independent HIV env having the nucleotide sequence of SEQ ID NO:4.

[0013] The invention includes an isolated nucleic acid comprising aportion of a HIV-1 env gene which confers CD4 independence on at leastone HIV-1 env clone.

[0014] The invention further includes a chimeric nucleic acid comprisinga first portion and a second portion, the first portion encoding atleast a portion of an HIV-1/IIIBx 8x env coding sequence and the secondportion encoding at least a portion of an HIV-1 env coding sequencewhich is not an 8x env.

[0015] In one aspect, the second portion is an env coding sequenceselected from the group consisting of an S10 env, an HXB2 env, a BaLenv, and an IIIB env.

[0016] In another aspect, the second portion comprises a chemokinereceptor binding site selected from the group consisting of a CXCR4chemokine receptor binding site, and a CCR5 chemokine receptor bindingsite.

[0017] In yet another aspect, the second portion comprises a V3-loopcoding sequence selected from the group consisting of a V3-loop for aCXCR4 chemokine receptor binding site, and a V3-loop for a CCR5chemokine receptor binding site.

[0018] The invention includes an isolated HIV-1 gp120 polypeptidecomprising a stably exposed chemokine coreceptor binding site.

[0019] The invention also includes an isolated polypeptide comprising anHIV-1/IIIBx 8x Env. In one aspect, the polypeptide shares at least about98% homology with SEQ ID NO:3.

[0020] In another aspect, the isolated polypeptide comprises the aminoacid sequence of SEQ ID NO:3.

[0021] The invention includes a chimeric HIV-1 Env polypeptidecomprising a gp120 polypeptide wherein the chimeric polypeptidecomprises a first portion comprising an HIV-1/IIIBx 8x gp120, thechimeric polypeptide further comprising a second portion comprising agp120 from an HIV-1 other than HIV-1/IIIBx 8x.

[0022] The invention further includes a chimeric HIV-1 Env polypeptidewherein the polypeptide is CD4-independent, and further wherein thepolypeptide comprises a chemokine receptor binding site selected fromthe group consisting of a CXCR4 chemokine receptor binding site, and aCCR5 chemokine receptor binding site.

[0023] In one aspect, the second portion comprises a V3-loop selectedfrom the group consisting of a HXB V3-loop, an 8x V3-loop, a BaLV3-loop, a YU-2 V3-loop, and an 89.6 V3-loop.

[0024] The invention includes a composition comprising a CD4-independentHIV-1 comprising a gp120 polypeptide comprising a stably exposedchemokine receptor binding site wherein the HIV-1 is more sensitive toantibody neutralization than an otherwise identical HIV-1 which does notcomprise a stably exposed chemokine receptor binding site.

[0025] The invention also includes a pharmaceutical compositioncomprising a CD4-independent HIV-1 Env protein, wherein the HIV-1 Envcomprises at least one mutation causing the chemokine coreceptor bindingsite to be stably exposed.

[0026] In one aspect, the HIV-1 Env is HIV-1/IIIBx 8x.

[0027] The invention includes a vaccine comprising an immunogenic doseof a CD4-independent HIV-1 Env.

[0028] In one aspect, the HIV- 1 Env is selected from the groupconsisting of a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1Env, and a cell expressing HIV-1 Env.

[0029] The invention includes a vector comprising an isolated nucleicacid encoding a CD4-independent human HIV-1 env, or a mutant,derivative, or fragment thereof.

[0030] The invention also includes a vector comprising an isolatednucleic acid comprising a portion of a HIV-1 env gene which confers CD4independence on at least one HIV-1 env clone.

[0031] The invention includes a vector comprising a chimeric nucleicacid comprising a first portion and a second portion, the first portionencoding at least a portion of an HIV-1/IIIBx 8x env coding sequence andthe second portion encoding at least a portion of an HIV-1 env codingsequence which is not an 8x env.

[0032] The invention includes a cell comprising an isolated nucleic acidencoding a CD4-independent human HIV-1 env, or a mutant, derivative, orfragment thereof.

[0033] The invention also includes a cell comprising an isolated nucleicacid comprising a portion of a HIV-1 env gene which confers CD4independence on at least one HIV-1 env clone.

[0034] The invention further includes a cell comprising a chimericnucleic acid comprising a first portion and a second portion, the firstportion encoding at least a portion of an HIV-1/IIIBx 8x env codingsequence and the second portion encoding at least a portion of an HIV-1env coding sequence which is not an 8x env.

[0035] The invention includes a cell comprising an isolated HIV-1 gp120polypeptide comprising a stably exposed chemokine receptor binding site.

[0036] The invention also includes a cell comprising an isolatedpolypeptide comprising an HIV-1/IIIBx 8x Env.

[0037] The invention includes a cell comprising a chimeric HIV-1 Envpolypeptide comprising a gp120 polypeptide wherein the chimericpolypeptide comprises a first portion comprising an HIV-1/IIIBx 8xgp120, the chimeric polypeptide further comprising a second portioncomprising a gp120 from an HIV-1 other than HIV-1/IIIBx 8x.

[0038] The invention also includes a cell comprising chimeric HIV-1 Envpolypeptide wherein the polypeptide is CD4-independent, and furtherwherein the polypeptide comprises a chemokine receptor binding siteselected from the group consisting of a CXCR4 chemokine receptor bindingsite, and a CCR5 chemokine receptor binding site.

[0039] In one aspect, the second portion comprises a V3-loop selectedfrom the group consisting of a HXB V3-loop, an 8x V3-loop, a BaLV3-loop, a YU-2 V3-loop, and an 89.6 V3-loop.

[0040] The invention includes a cell comprising a composition comprisinga CD4-independent HIV-1 Env comprising a gp120 polypeptide comprising astably exposed chemokine receptor binding site wherein the HIV-1 is moresensitive to antibody neutralization than an otherwise identical HIV-1which does not comprise a stably exposed chemokine receptor bindingsite.

[0041] The invention includes a method of identifying an amino acidresidue of an HIV-1 Env protein which is involved in CD4 independence.The method comprises obtaining a full-length env coding sequence from anEnv clone which is CD4-independent and replacing at least a portion ofthe said env coding sequence with a coding sequence from an Env clonewhich is CD4-dependent to form a chimera, wherein when the chimera isCD4-dependent it is an indication that the portion of the env codingsequence is involved in CD4-independence, thereby identifying an aminoacid residue involved in CD4-independence.

[0042] The invention also includes a method of eliciting an immuneresponse to a HIV-1 chemokine receptor binding site in a mammal. Themethod comprises administering an immunogenic dose of a CD4-independentHIV-1 Env protein to a mammal, wherein the protein comprises a stablyexposed chemokine receptor binding site, thereby eliciting an immuneresponse to a HIV-1 chemokine receptor binding site in a mammal.

[0043] The invention also includes a method of identifying a compoundwhich affects exposure of an HIV-1 gp120 chemokine receptor bindingsite. The method comprises contacting a cell with the compound prior toor contemporaneous with contacting the cell with a labeled gp120 with orwithout pre-incubation of the gp120 with soluble CD4, measuring theamount of label bound to the cell, and comparing the amount of labelbound to the cells contacted with the compound to the amount of labelbound to otherwise identical cells not contacted with the compound,wherein a higher or lower amount of label bound to the cells contactedwith the compound compared with the amount of label bound to theotherwise identical cells not contacted with the compound, is anindication that the compound affects exposure of an HIV-1 gp120chemokine receptor binding site.

[0044] The invention includes a method of identifying a small-moleculewhich inhibits binding of an HIV-1 gp120, using its chemokine receptorbinding site, to a chemokine receptor. The method comprises contacting acell with the molecule prior to or contemporaneous with contacting thecell with labeled gp120 with or without pre-incubation of said gp120with soluble CD4, measuring the amount of label bound to the cell, andcomparing the amount of label bound to the cell contacted with themolecule with the amount of label bound to an otherwise identical cellnot contacted with the molecule, wherein a lower amount of label boundto the cell contacted with the molecule compared with the amount oflabel bound to the otherwise identical cell not contacted with themolecule, is an indication that the molecule inhibits binding of anHIV-1 gp120 using its chemokine receptor binding site to a chemokinereceptor.

[0045] The invention includes a method of producing a CD4-independentchimeric HIV-1 Env clone comprising a variable chemokine receptorbinding site. The method comprises replacing the hypervariable V3-loopof the CD4-independent Env clone with the V3 loop of another HIV-1,wherein the V3-loop of another HIV-1 comprises a different chemokinereceptor binding site than that of the CD4-independent Env clone.

[0046] In one aspect, the CD4-independent clone is selected from thegroup consisting of HIV-1/IIIBx, and HIV-1/IIIBx 8x.

[0047] In another aspect, the V3-loop from another HIV-1 is selectedfrom the group consisting of a V3-loop from HIV-1/BaL, a V3-loop fromHIV-1/YU-2, a V3-loop from HIV-1/ADA, and a V3-loop from HIV-1/89.6.

[0048] The invention also includes a method of inhibiting HIV-1 gp120binding, using its chemokine receptor binding site, to a chemokinereceptor. The method comprises contacting said gp120 with asmall-molecule identified by a method of identifying a compound whichaffects exposure of an HIV-1 gp120 chemokine receptor binding site, themethod comprising contacting a cell with the compound prior to orcontemporaneous with contacting the cell with a labeled gp120 with orwithout pre-incubation of the gp120 with soluble CD4, measuring theamount of label bound to the cell, and comparing the amount of labelbound to the cells contacted with the compound to the amount of labelbound to otherwise identical cells not contacted with the compound,wherein a higher or lower amount of label bound to the cells contactedwith the compound compared with the amount of label bound to theotherwise identical cells not contacted with the compound, is anindication that the compound affects exposure of an HIV-1 gp120chemokine receptor binding site, thereby inhibiting HIV-1 gp120 binding,using its chemokine receptor binding site, to a chemokine receptor.

[0049] The invention includes a method of inhibiting HIV-1 infection ofa cell. The method comprises contacting the cell with a small-moleculewhich inhibits binding of an HIV-1 gp120 using its chemokine receptorbinding site to a chemokine receptor, wherein the small-molecule isidentified using a method of identifying a small-molecule which inhibitsbinding of an HIV-1 gp120, using its chemokine receptor binding site, toa chemokine receptor, the method comprising contacting a cell with themolecule prior to or contemporaneous with contacting the cell withlabeled gp120 with or without pre-incubation of said gp120 with solubleCD4, measuring the amount of label bound to the cell, and comparing theamount of label bound to the cell contacted with the molecule with theamount of label bound to an otherwise identical cell not contacted withthe molecule, wherein a lower amount of label bound to the cellcontacted with the molecule compared with the amount of label bound tothe otherwise identical cell not contacted with the molecule, is anindication that the molecule inhibits binding of an HIV-1 gp120 usingits chemokine receptor binding site to a chemokine receptor, therebyinhibiting HIV-1 infection of a cell.

[0050] The invention includes a composition comprising a CD4-independentHIV-1 Env and at least one compound used to treat HIV infection in apharmaceutically suitable carrier.

[0051] In one aspect, the HIV-1 Env is selected from the groupconsisting of a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1Env, and a cell expressing HIV-1 env.

[0052] In another aspect, the compound used to treat HIV infection isselected from the group consisting of a protease inhibitor, a reversetranscriptase nucleoside analog inhibitor, a reverse transcriptasenon-nucleoside analog inhibitor, an interferon, AZT, interleukin-2, anda cytokine.

[0053] The invention includes a method of treating HIV-1 infection in ahuman. The method comprises administering an immunogenic dose of aCD4-independent HIV-1 Env to an HIV-1 infected human, thereby treatingHIV-1 infection in the human.

[0054] In one aspect, the HIV-1 Env is selected from the groupconsisting of a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1Env, and a cell expressing HIV-1 env.

[0055] In another aspect, the method further comprises administering acompound used to treat HIV infection.

[0056] In yet another aspect, the compound used to treat HIV infectionis selected from the group consisting of a protease inhibitor, a reversetranscriptase nucleoside analog inhibitor, a reverse transcriptasenon-nucleoside analog inhibitor, an interferon, AZT, interleukin-2, anda cytokine.

[0057] In a further aspect, the compound is administered to said humanbefore, during or after administration of said CD4-independent HIV-1Env.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0058]FIG. 1A is a graph, comprising two panels, depicting viralreplication in CD4-positive and CD4-negative T cells by HIV-1/IIIB orHIV-1/IIIBx. CD4-negative BC7 cells (top panel) and SupT1 CD4-positivecells (bottom panel) were inoculated with equal amounts of HIV-1/IIIB(open diamonds) or HIV-1/IIIBx (solid circles) and viral replication wasdetermined by reverse transcriptase (RT) activity in culturesupernatants.

[0059]FIG. 1B is a graph depicting the inhibition of HIV-1/IIIBxreplication by anti-CXCR4 antibody (12G5). CD4-negative BC7 cells wereinoculated with HIV-1/IIIBx in the absence (open bars) or presence (5μg/ml, hatched bars, or 20 μg/ml, solid bars) of anti-CXCR4 antibody12G5 and reverse transcriptase (RT) activity was determined at the timepoints indicated.

[0060]FIG. 1C is an image, comprising two panels, depicting cell fusioninduced by HIV-1/IIIBx on murine cells expressing CXCR4.HIV-1/IIIBx-infected BC7 cells were co-cultured with murine 3T3 cellswhich do not express CXCR4 (3T3, left panel) or with 3T3 cells thatexpress human CXCR4 (3T3/CXCR4, right panel) for 24 hours and then thecells were stained for syncytial formation as described in Endres et al.(1996, Cell 87:745-756).

[0061]FIG. 2 is a graph depicting the fusion activity, expressed inrelative light units (RLUs), of IIIBx env genes. The env genes indicatedwere cloned into pSP73, transfected into QT6 cells, and the genes wereevaluated in fusion assays on QT6 cells expressing CD4 plus CXCR4, CXCR4alone, or CD4 alone as described in Rucker et al. (1997, MethodsEnzymol. 288:118-133) and as described elsewhere herein. The results areexpressed as the mean +SEM in RLU normalized to the activity of 8x onCXCR4+/CD4+ cells. Also shown are the fusion activities for 8x and HXBc2Envs containing a D368R mutation that ablates the CD4-binding site asdescribed in Olshevsky et al. (1990, J. Virol. 64:5701-5705).

[0062]FIG. 3A is a graph demonstrating the fusion activity of theHIV-1/IIIBx env gene. The 8x env was inserted into pNL4-3 and a viralstock was generated after transfection of BC7 cells. Equal amounts ofthe resulting virus (designated NL43/8x) and HIV-1/IIIB were inoculatedonto SupT1 and BC7 cells and RT levels were monitored over time.

[0063]FIG. 3B is an image of a Western blot depicting the evaluation ofthe size of TM polypeptide of various viruses. Viral lysates fromHIV-1/IIIB infected SupT1 cells (lane 1), IIIBx infected BC7 cells (lane2), and NL43/8x-infected BC7 cells were evaluated by Western blot usinganti-TM mouse monoclonal antibody D12. Consistent with the sequenceanalyses (FIG. 4), both IIIBx and NL43/8x exhibited a truncated TMprotein.

[0064]FIG. 4 is a diagram depicting the amino acid sequence analysis ofIIIBx env clones. Sequence analysis for IIIBx env clones 8x (SEQ IDNO:3) and S10 (SEQ ID NO:12) are compared with that of HXBc2 (SEQ IDNO:11). The shaded regions indicate mutations that are also found inother clones from HIV-1/IIIB. The predicted N-linked glycosylation sitesare indicated by the shaded gray circle symbol directly over the aminoacid position (where amino acids are designated using a one-lettercode). The positions of the variable loops, the gp120/gp41 cleavage siteand the TM membrane spanning domain (msd) are also indicated above theamino acid sequence of HXBc2. The 8x sequence contains a frame shiftmutation at amino acid position 706 which results in a prematurelytruncated cytoplasmic tail compared with HxBc2. S10 contains a deletionof 50 nucleotides which also leads to a frameshift and a prematurelytruncated cytoplasmic tail. In FIG. 4, dashes indicate amino acidresidues that are identical to the corresponding amino acid residue ofHXBc2.

[0065]FIG. 5 is a diagram depicting the evaluation of chimeric Envproteins in fusion assays. The diagram depicts the env genes from 8x,S10, HXBc2, and chimeras constructed using the indicated restrictionsites shown at the top of the diagram. The mutations present in 8x areindicated above the top schematic. The chimeras were cloned into pSP73and evaluated in cell fusion assays as described in FIG. 6, infra.

[0066]FIG. 6 is a graph depicting the evaluation of chimeric Envproteins in fusion assays. The chimeric Env proteins constructed betweenHXBc2 and 8x which are shown in FIG. 5, supra, were evaluated in fusionassays on QT6 target cells expressing CXCR4 alone, CXCR4 and CD4, or CD4alone. The results are expressed as luciferase activity relative to thatof HXBc2 on CXCR4+g/CD4+ cells (i.e., relative luciferase units, RLU).The bars indicate the mean RLU for 3 experiments+SEM.

[0067]FIG. 7 is a graph depicting the mapping of determinants for aCD4-dependent clone of IIIBx. The fusion activity is shown for theCD4-dependent S10 clone of IIIBx and for S10/8x chimeras as indicated inFIG. 5, supra. In addition, the activity is shown for an S10 Env inwhich the G431E mutation in the C4 domain was corrected (S10-E431G ) andfor an 8x Env that contained this mutation (8x-G431E). The results areexpressed as the percentage of 8x luciferase activity on target cellsthat coexpressed CXCR4 and CD4.

[0068]FIG. 8 is graph depicting the CCR5 tropism of Env proteinscontaining V3 loop from the CCR5-tropic Env, HIV-1/BaL. HXB2 (amolecular clone derived from the IIIB swarm) Env, which isCD4-dependent, and 8x Env proteins containing the V3 loop from HIV-1/BaLwere constructed and their fusion activity was compared to the parentalHBXc2 or 8x Envs on target cells that expressed CCR5 or CXCR4±CD4.Fusion activity is expressed as the percentage of luciferase activity ofHxBc2 on target cells that expressed both CXCR4 and CD4. The barsindicate the mean+SEM.

[0069]FIG. 9A is an image of a space-filling model depicting theHIV-1/HXB2 gp120 core crystal structure and demonstrating the locationof HIV-1/IIIBx mutations on the gp120 crystal structure. The corecrystal structure is depicted in white in conjunction with a ribbondiagram of CD4 (Kwong et al., 1998, Nature 393:648-659) which is shownin the bottom right quadrant of the image. The amino acid sites at whichmutations produced a 50% decrease or increase in gp120 binding to CCR5(Rizzuto et al., 1998, Science 280:1949-1953) are shown in red. Withoutwishing to be bound by theory, of the 6 mutations in 8x that could bemapped onto the gp120 core, 3 (shown in light blue) are locatedimmediately adjacent to this putative chemokine receptor binding site.

[0070]FIG. 9B is an image of a ribbon diagram of the gp120/CD4 complexdepicted in a slightly different orientation from that shown in FIG. 9A,supra, in order to indicate the position of the G431E mutation, whichwas sufficient to abrogate CD4-independence but not CD4-dependent fusionof the 8x clone.

[0071]FIG. 10A is a graph depicting CD4-independent cell-cell fusion byHXB or 8x Env clones. QT6 effector cells expressing HXB or 8x Env, asindicated, as well as T7 polymerase were mixed with QT6 target cellsexpressing chemokine receptor CXCR4 (cross-hatched bars), CD4 (openbars), or CXCR4/CD4 (closed bars) and the luciferase gene under controlof the T7 promoter. HIV-1/IIIB is an uncloned virus from which severalmolecular clones, such as HXBc2 (“HXB”) and IIIBx (“8x”), have beenderived. The data disclosed herein compare these two Env molecularclones. Luciferase is produced in this assay only if Env mediates fusionbetween effector and target cells. The results for each Env areexpressed in RLUs and are normalized to the amount of fusion obtainedwith IIIB Env effector and CXCR4/CD4 target cells. The results of atypical experiment are shown.

[0072]FIG. 10B is a graph depicting CD4-independent cell-cell fusion byHXB-V3BaL or 8x-V3BaL Env clones. Luciferase reporter viruses bearingHXB-V3BaL or 8x-V3BaL Env proteins, as indicated, were used to infect293T cells expressing CCR5 (cross-hatched bars), CD4 (open bars), orCCR5/CD4 (gray bars) and the luciferase gene under control of the T7promoter. The amount of luciferase activity was determined 3 days afterinfection. The results for each Env are expressed in RLU and arenormalized to the results obtained with virions bearing the IIIB-BaL Envand CCR5/CD4 target cells. The results of a typical experiment areshown.

[0073]FIG. 11 is a graph depicting cell-surface binding of variousgp120s in cells expressing CD4, CXCR4 or CCR5. Radioiodinated gp120swere incubated with 293T cells transiently transfected with coreceptoror CD4 plasmids. Soluble CD4 (sCD4) was added to the binding reaction asindicated. The amount of specific radioactivity bound to the cells ispresented and is normalized for each gp120 indicated such that bindingto CD4 represents 100%. Each value represents the average of ≧3independent experiments and the error bars represent SEMs. The followingcombinations are shown: cells expressing empty vector pCDNA3 (openbars), cells expressing CD4 (solid bars), cells expressing CXCR4 (darkgray bars), cells expressing CXCR4 with sCD4 added (dark cross-hatchbars), cells expressing CCR5 (light gray bars), cells expressing CCR5with sCD4 added (light cross-hatch bars).

[0074]FIG. 12 is an image of a space-filling model of gp120 bound to CD4depicting the overlap between the CCR5 coreceptor binding site and theMAb 17b epitope. The amino acid residues shown by Rizzuto et al. (1998,Science 280:1949-1953), to decrease CCR5 binding by greater than 50%when mutated while reducing CD4 binding by less than 50% are shown inred. The contact residues for MAb 17b are shown in light blue, and theresidues involved in both CCR5 and 17b binding are shown in lavender.Three residues that differ between 8x and IIIB in the vicinity of thecoreceptor binding site as disclosed previously in Example 1 and whichmay impact CD4-independence, are shown in green. One of these residues,423, is also a contact site for MAb 17b. The stems of the hypervariableV1/V2 and V3 loops are shown in orange.

[0075]FIG. 13 is a graph depicting the sensorgrams for gp120 binding tothe CD4i MAb 17b. MAb 17b was attached to the sensor surface after whichthe indicated gp120 molecule (at equal concentrations), with or withoutprior incubation with saturating levels of sCD4 as indicated, wereapplied to the flow cell. A 300 second association was followed by awash with running buffer for an additional 300 seconds during whichdissociation was measured. The kinetic constants derived from lineartransformations of the data are presented in Table 1 elsewhere herein.

[0076]FIG. 14, comprising FIGS. 14A and 14B, lists the nucleotidesequence of env obtained from clone 8x.

DETAILED DESCRIPTION OF THE INVENTION

[0077] The invention is based on the discovery of a CD4-independentvariant of HIV-1/IIIB, designated HIV-1/IIIBx (IIIBx), and a functionalfull-length env clone therefrom termed HIV-1/IIIBx.8 (8x), which allowthe study of the mechanism for virus infection of host cells involvingcell receptor proteins. Further, the present invention relates to theconstruction of chimeras comprising portions of a nucleic acid encoding8x env covalently linked to a least one nucleic acid encoding a portionof an env from another HIV-1 virus. Thus, the chimeras are produced bycombining portions of the 8x env coding sequence with portions of theenv coding sequences of other virions leading to the further discoveryof which portion(s) of the 8x HIV-1 env sequence is involved inCD4-independence.

[0078] CD4-independence is important in that it is an indicator that thechemokine binding site of gp120 is stably exposed on the virus envelopeand is capable of binding to the cellular chemokine receptor bindingprotein without prior binding of the gp120 to CD4. Typically, thechemokine binding site is hidden until such binding to CD4 causes aconformational change exposing the site and resulting in a “triggered”conformation capable of binding to the chemokine receptor protein on thehost cell. Therefore, the CD4-independent gp120 represents a stableintermediate configuration which may be used to, inter alia, identifythe protein determinants involved in gp120 binding to a chemokinereceptor protein, produce neutralizing antibodies capable of recognizingthe gp120 chemokine receptor binding site, and to identifysmall-molecule inhibitors which can block gp120/chemokine receptorbinding.

[0079] Accordingly, understanding which portions of the Env are involvedin virus binding to cell proteins and thereby mapping the proteindeterminants involved in HIV-1 virus binding to host cell receptors isimportant in the development of effective antiviral vaccines to viralprotein domains crucial for virus infection. Such domains are believedto be highly conserved but somehow “camouflaged” from the immune systemsuch that a protective immune response is not mounted to such proteindomains. Therefore, identification of these protein domains and theability to present them to the immune system such that an immuneresponse is generated to HIV-1 is an important goal of vaccinedevelopment to this important human pathogen.

[0080] Moreover, production of chimeras has led to the discovery thatthe CD4 dependence trait and the choice of chemokine receptor arefunctionally dissociable traits. One skilled in the art wouldappreciate, based upon the disclosure provided herein, that suchchimeras are useful for mapping the various structural and functionalelements of the nucleic acid encoding env and the Env protein encodedthereby. Thus, by combining various portions of different viruses havingdifferent properties, e.g., CD4-dependence or independence and/ordifferent affinities for various chemokine receptors, the variousfunctional elements of the Env protein may be examined and identified.

[0081] In one embodiment, replacing the V3-loop portion of 8x gp120,which binds the CXCR4 chemokine receptor in the absence of CD4, with theV3-loop of HIV-1/BaL, which is a virus strain that is CD4-dependent andbinds the CCR5 chemokine coreceptor, converts the chimeric gp1208x/V3-BaL to a CCR5 binding protein which retains CD4-independence. Thisfurther demonstrates that CD4-independence exposes the chemokinereceptor binding domain such that the preceding step of CD4-binding bygp120 is no longer required regardless of the choice of chemokinereceptor. These data also suggest that a chemokine receptor binding siteexists on the gp120 that is able to interact with genetically divergentchemokine receptors (i.e., CXCR4 and CCR5) and this site is functionaland likely exposed on CD4-independent viruses.

[0082] In addition, the present invention teaches that theCD4-independent gp120 protein exists in a stable partially “triggered”state, wherein the chemokine coreceptor binding site is more exposed inthe CD4-independent gp120 protein than in the CD4-dependent conformationof the HIV-1 gp120 molecule. This has the effect of rendering theCD4-independent virus more susceptible to neutralization by anti-HIV-1antibodies from mouse, human and rabbit. Therefore, the presentinvention has important implications for the development of HIV-1therapeutics since the availability of a stably exposed, highlyconserved chemokine receptor binding site, which may be otherwisecamouflaged to escape immune detection, should facilitate thedevelopment of a humoral and/or cellular immune response and ofsmall-molecule inhibitors to block this virus-host protein interaction,thereby preventing HIV-1 infection.

[0083] The present invention includes an isolated nucleic acid encodinga CD4-independent HIV env coding sequence which is comprised of twocomponents, a portion encoding gp120 and a portion encoding gp41. In oneembodiment, the full-length env clone of CD4-independent HIV-1/IIIBx,i.e., 8x, has been isolated (SEQ ID NO:3 and SEQ ID NO:4; see FIGS. 3and 14A and 14B, respectively). Further, the mutations in the 8x clonewere identified relative to the known env coding sequence of HXBc2(GenBank Accession No. AF038399) (SEQ ID NO:11) and are disclosed inFIG. 4. However, the present invention should not be construed to belimited to a full-length env clone of the CD4-independent HIV-1/IIIBxvariant. Rather, the present invention should be construed to encompasspartial env clones. Indeed, the data disclosed herein demonstrate thatthe entire env coding sequence of 8x is not required forCD4-independence. Thus, at least one mutation present in the 8x envcoding sequence confers CD4-independence to 8x, but not all mutations inthe clone are required for purposes of the present invention. Further,completely separate mutations of gp120 can also confer CD4-independence.

[0084] The experiments disclosed in the Examples below disclose theisolation of a CD4-independent strain of the invention, HIV-1/IIIBx,which was able to infect both CD4⁺ SupT1 cells and CD4⁻ BC7 cells, aSupT1 variant, as demonstrated by a reverse transcriptase activity assay(FIG. 1A). However, the present invention is not limited solely toinfection of BC7 or SupT1 cells by HIV-1. Rather, the “CD4-independence”of the present invention encompasses infection by HIV-1 of any cell typewhich does not express CD4. Further, as discussed previously herein, aCD4-independent HIV-1 strain may also infect cells that are CD4⁺although CD4/gp120 interaction is not required for infection of thesecells by the CD4-independent HIV-1. Moreover, a CD4-independent HIV-1strain need not infect every CD4⁻ cell type. Rather, the HIV-1 strainneed only be able to infect at least one CD4⁻ cell type while itsotherwise identical parental strain from which the clone was obtainedcannot infect that cell type.

[0085] Additionally, for purposes of the invention, an HIV-1 strainvariant is considered CD4-independent when it is able to infect at leastabout 5% of the susceptible cells in culture or the level of infectionis about two to three-fold compared to background levels.

[0086] It will be appreciated by one skilled in the art, based upon thedisclosure provided herein, that a CD4-independent isolate of an HIV-1strain may be obtained by passaging a CD4-dependent HIV-1 swarminitially grown in CD4₊ cells onto cells which are CD4⁻. As disclosed inthe experiments described in Example 1 herein, HIV-1/IIIBx was obtainedby passaging virus in CD4⁺ SupT1 cells followed by passaging virus onthe otherwise identical but CD4⁻ BC7 cells. However, the inventionshould not be construed to be limited to these particular cell types.Instead, the invention encompasses a variety of CD4⁺ and CD4⁻ cellsincluding, but not limited to, 293, Cf2TH, CCC⁺L⁻, and QT6 cells as wellas stably transfected cells (U87, HeLa, HOS) that express a recombinantchemokine receptor in the presence or absence of CD4.

[0087] In other related aspects, the invention includes vectors whichcontain such an isolated nucleic acid comprising at least a portion ofthe HIV-1 env and which isolated nucleic acid is preferably capable ofdirecting expression of the protein encoded by the nucleic acid; andvirions, proviruses, and/or cells containing such vectors.

[0088] As the present experimental examples demonstrate, the nucleicacid encoding the Env protein may be cloned into various plasmidvectors. However, the present invention should not be construed to belimited to these plasmids or to any particular vector. Instead, thepresent invention should be construed as encompassing a wide plethora ofvectors which are readily available and/or well-known in the art.Therefore, although in one embodiment, the full-length env codingregions were amplified by PCR and cloned into the plasmid pCDNA3, andthe inserts were then sub-cloned into the 3′ hemigenome of pNL4-3, thepresent invention should not be construed to be limited to these, or toany other, specific vectors.

[0089] The isolated nucleic acid of the invention should be construed toinclude an RNA or a DNA sequence encoding an Env protein of theinvention, and any modified forms thereof, including chemicalmodifications of the DNA or RNA which render the nucleotide sequencemore stable when it is cell free or when it is associated with a cell.Chemical modifications of nucleotides may also be used to enhance theefficiency with which a nucleotide sequence is taken up by a cell or theefficiency with which it is expressed in a cell. Any and allcombinations of modifications of the nucleotide sequences arecontemplated in the present invention.

[0090] The present invention also includes an isolated polypeptidecomprising the amino acid sequence of HIV-1/IIIBx 8x.

[0091] The present invention also provides for analogs of proteins orpeptides which comprise a gp120 protein as disclosed herein. Analogs maydiffer from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both. For example, conservative amino acid changes maybe made, which although they alter the primary sequence of the proteinor peptide, do not normally alter its function. Conservative amino acidsubstitutions typically include substitutions within the followinggroups:

[0092] glycine, alanine;

[0093] valine, isoleucine, leucine;

[0094] aspartic acid, glutamic acid;

[0095] asparagine, glutamine;

[0096] serine, threonine;

[0097] lysine, arginine;

[0098] phenylalanine, tyrosine.

[0099] Modifications (which do not normally alter primary sequence)include in vivo, or in vitro, chemical derivatization of polypeptides,e.g., acetylation, carboxylation, or biotinylation. Also included aremodifications of glycosylation, e.g., those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps; e.g., by exposing thepolypeptide to enzymes which affect glycosylation, e.g., mammalianglycosylating or deglycosylating enzymes. Also embraced are sequenceswhich have phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

[0100] Also included are polypeptides which have been modified usingordinary molecular biological techniques so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

[0101] Further, the invention should be construed to include naturallyoccurring variants or recombinantly derived mutants of HIV-1/IIIBx 8xenv sequences, which variants or mutants render the protein encodedthereby either more, less, or just as biologically active as thefull-length 8x env clone of the invention.

[0102] In addition, the present invention includes mutants or variantsof 8x gp120 comprising an altered chemokine receptor binding site. Asdiscussed previously elsewhere herein, the gp120 protein comprises achemokine receptor binding domain which mediates gp120 binding tovarious cellular chemokine receptor proteins, which binding typicallyoccurs after gp120 binding to CD4. As disclosed in the experimentalresults which follow this section, 8x gp120 binds to CXCR4 chemokinereceptor and does not require binding to CD4 before doing so. Further,the data disclosed elsewhere herein demonstrate that introduction of aportion of a nucleic acid encoding a portion of an HIV-1/BaL gp120 intothe coding sequence of 8x gp120 gives rise to a chimeric protein that nolonger binds to CXCR4. Instead, the chimeric gp120 now binds CCR5. Suchmutants are useful in the methods of the invention for the study of therole of gp120-chemokine receptor protein interaction in HIV-1 virusinfection. The present invention should not be construed to be limitedsolely to a chimeric gp120 wherein a portion of the nucleic acidencoding 8x gp120 has been replaced a portion of a nucleic acid encodingBaL gp120. Instead, the present invention should be construed to includeother chimeras wherein any portion or portions of the nucleic acidencoding 8x gp120 may be replaced by at least one portion of a nucleicacid encoding a gp120 from any other HIV-1 strain, preferably, thosestrains of HIV (or SIV) that use CCR5 as a coreceptor. Further, suchportions should not be construed as being limited to any particulardomain of gp120, but rather, the portion of gp120 substituted may befrom any portion of the sequence encoding the protein. Therefore, theresulting chimeric nucleic acid and the protein expressed therefrom maybe a chimera comprised of various gp120s from several HIV-1 strains, inany combination possible.

[0103] As more specifically set forth elsewhere herein, a mutant gp120gene which encodes a gp120 protein comprising an insertion, deletion, orsubstitution, whereby amino acids residues at or near the putativechemokine receptor binding site are altered, or whereby a truncatedcytoplasmic tail of Env is produced, is useful in studying theassociation of gp120 with a host cell chemokine receptor protein.Indeed, as disclosed in the experiments described below, several suchmutants have been discovered herein (see Table 1 and FIG. 3). However,the invention should not be construed as being limited to only thesemutants; rather, the invention encompasses other mutants, comprisingdeletion, substitution, and point mutations, which demonstrate alteredbinding to chemokine receptor protein compared with the wild type gp120and which mutants demonstrate CD4-independence.

[0104] The invention should also be construed to include DNA encodingvariants of HIV-1 Env which may or may not retain biological activity.Such variants, i.e., analogs of proteins or polypeptides of gp120, gp41(also referred to as TM), include proteins or polypeptides which havebeen or may be modified using recombinant DNA technology such that theprotein or polypeptide possesses additional properties which enhance itssuitability for use in the methods described herein, for example, butnot limited to, variants conferring enhanced stability of the exposedchemokine receptor binding site, enhanced specific binding to CD4,CXCR4, CCR5, and the like.

[0105] The present invention includes analogs of the 8x Env protein.Analogs can differ from naturally occurring proteins or peptides byconservative amino acid sequence differences or by modifications whichdo not affect sequence, or by both. For example, conservative amino acidchanges may be made, which although they alter the primary sequence ofthe protein or peptide, do not normally alter its function.

[0106] Preferably, the amino acid sequence of an 8x Env analog is about70% homologous, more preferably about 80% homologous, even morepreferably about 90% homologous, more preferably, about 95% homologous,and most preferably, at least about 99% homologous to the amino acidsequence of 8x env (SEQ ID NO:3) disclosed herein at FIG. 4.

[0107] The invention should not be construed as being limited solely tothe DNA and amino acid sequences disclosed herein. Once armed with thepresent invention, it is readily apparent to one skilled in the art thatother CD4-independent env clones of HIV-1 may be obtained by followingthe procedures described herein in the experimental details section forthe isolation of the 8x env nucleic acid (SEQ ID NO:4) encodingCD4-independent Env disclosed herein.

[0108] The invention should therefore be construed to include any andall nucleic acid sequences encoding HIV-1/IIIBx 8x Env and amino acidsequences having substantial homology to the nucleic acid encoding 8xenv disclosed herein (SEQ ID NO:4) and the amino acid sequence (SEQ IDNO:3) shown in FIG. 4. Preferably, DNA which is substantially homologousis about 50% homologous, more preferably about 70% homologous, even morepreferably about 80% homologous and most preferably about 90% homologousto the 8x env sequence (SEQ ID NO:4) disclosed herein. Preferably, anamino acid sequence which is substantially homologous is about 50%homologous, more preferably about 70% homologous, even more preferablyabout 80% homologous and most preferably about 90% homologous to the 8xEnv amino acid sequences (SEQ ID NO:3) shown in FIG. 4.

[0109] Any number of procedures may be used for the generation of mutantor variant forms of 8x env. For example, generation of mutant forms of8x which are not CD4 independent was accomplished herein by introducingportions of a nucleic acid encoding env from a virus which wasCD4-dependent using recombinant DNA methodology well known in the artsuch as, for example, as described in Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork) and Ausubel et al. (1997, Current Protocols in Molecular Biology,Green & Wiley, New York). Mutant Env so generated is expressed and theresulting protein is assessed for its ability to bind CD4 in a real timebiosensor assay such as that described herein. Mutant proteins whichbind chemokine receptor protein in a CD4-independent manner were thenexamined by RT, fusion activity, real time binding/dissociationkinetics, and other such assays.

[0110] Procedures for the introduction of amino acid changes in aprotein or polypeptide by altering the DNA sequence encoding thepolypeptide are well known in the art and are also described in Sambrooket al. (1989, supra); Ausubel et al. (1997, supra).

[0111] The invention also includes an isolated nucleic acid havingnucleic acid sequence which is complementary to a portion or all of thenucleic acid encoding HIV-1 Env (SEQ ID NO:4).

[0112] As used herein, the term “fragment” as applied to a nucleic acid,may ordinarily be at least about 100 nucleotides in length, typically,at least about 200 nucleotides, more typically, from about 300 to about600 nucleotides, typically at least about 700 to about 1000 nucleotides,preferably at least about 1000 to about 1400 nucleotides, even morepreferably at least about 1600 nucleotides to about 2000 nucleotides,and most preferably, the nucleic acid fragment will be greater thanabout 2400 nucleotides in length.

[0113] The invention further includes a cell comprising the nucleicacids of interest. The nucleic acids need not be integrated into thecell genome nor do they need to be expressed in the cell. Moreover, thecell may be a prokaryotic or a eukaryotic cell and the invention shouldnot be construed to be limited to any particular cell line or type.

[0114] The invention also includes antibodies specific for the chemokinereceptor binding site of gp120, or a portion thereof, which antibodiescomprise a monoclonal antibody.

[0115] In one embodiment, the antibody is a murine monoclonal antibodyto gp120 (17b) the epitope of which overlaps with the chemokine receptorbinding site, as well as a murine monoclonal antibody to gp120 termed48d (Thali et al., 1993, J. Virol. 67:3978-3988). However, the inventionshould not be construed as being limited solely to these antibodies butrather, should be construed to include other antibodies, as that term isdefmed herein, to Env, or portions thereof, which antibodies perform ina manner substantially similar to those described herein in that, interalia, the antibodies bind to gp120 chemokine receptor binding site, andthey are able to inhibit HIV-1 infection as measured by RT activity andcell fusion activity.

[0116] The invention also comprises an isolated polypeptide comprisingthe amino acid sequence of 8x Env protein, and mutants, variants andfragments thereof.

[0117] The peptides of the invention may be substantially pure. Asubstantially pure peptide is purified by following known procedures forprotein purification, wherein an immunological, enzymatic or other assayis used to monitor purification at each stage in the procedure. Proteinpurification methods are well known in the art, and are described, forexample in Deutscher et al. (1990, In: Guide to Protein Purification,Harcourt Brace Jovanovich, San Diego).

[0118] The invention should thus be construed to include nucleic acidencoding desired proteins and fragments of nucleic acid encoding desiredpolypeptides.

[0119] The present invention includes an isolated nucleic acid encodinga chimeric protein comprising a first portion and a second portion. Inone embodiment, the chimeric nucleic acid comprises a first portionencoding 8x env and a second portion encoding an env from S10, IIIB, orHXB2. Although these chimeras were useful in mapping which regions of 8xare required for CD4-independence, the present invention should not beconstrued to be limited to these chimeras. Rather, the invention shouldbe construed to encompass any chimeras in the env coding region whichmay be constructed comprising any portion of 8x and any HIV-1 virusstrain or variant thereof.

[0120] Further, in another embodiment, the chimeras comprised a portionof the 8x env coding region and a portion of the env coding region of aCCR5-tropic HIV-1 strain, BaL. More'specifically, the embodimentcomprises the 8x env clone with the nucleic acid portion encoding theV3-loop of BaL. However, the present invention should not be construedto be limited to this particular portion of the env coding region or tothis particular strain of HIV-1. Rather, as previously discussedelsewhere herein, the present invention includes the substitution of anyportion of the 8x env coding sequence with a portion of the env codingsequence of at least one other HIV-1 strain or variant, and any possiblepermutation thereof. Therefore, the chimeras, both nucleic acid andamino acid expressed therefrom, include combinations from two or moreHIV-1 env coding regions of interest. Thus, armed with the disclosureprovided herein, the production of an almost infinite combination ofchimeras with the predicted effects disclosed herein would be clear toone skilled in the art.

[0121] The invention also includes a method of identifying an amino acidresidue of an HIV-1 Env protein which is involved in CD4-independence.The method comprises producing chimeric proteins comprising at least aportion from a CD4-independent Env clone and at least a second portionfrom a CD4-dependent Env clone. The resulting chimera is then examinedto determine the ability of the chimeric protein to mediateCD4-independent infection by various assays as disclosed elsewhereherein. As discussed previously herein, a preferred embodiment isdisclosed wherein portions of the 8x env coding sequence were combinedwith various portions of the env coding sequences of severalCD4-dependent HIV-1 strains, e.g., S10 and HxBc2. Also as notedpreviously herein, the present invention is not limited to theseparticular combinations or to these particular strains. Rather, oneskilled in the art would appreciate, based on the disclosure providedherein, that any combination of CD4-dependent and -independent envcoding sequences may be examined to map the CD4-independentdeterminants. Further, the CD4-independence may be examined using avariety of assays on various mammalian cell lines also as describedpreviously elsewhere herein.

[0122] The present invention also includes an isolated gp120 proteincomprising a stably exposed chemokine receptor binding site. In oneembodiment, the increased exposure of the chemokine receptor bindingsite was determined by measuring the real time binding kinetics of thevarious proteins in biosensor experiments and the enhancedneutralization of the virus by anti-HIV antibodies and bycrystallographic analyses. However, the present invention should not beconstrued to be limited to these particular assays. Rather, other assayswell-known in the art or to be developed for the study ofprotein-protein interactions may be used to measure the exposure of thechemokine receptor binding site of a gp120 or Env protein of interest.

[0123] The invention includes a method of eliciting an immune responseto a HIV-1 chemokine receptor binding site. The method comprisesadministering an immunogenic dose of a CD4-independent HIV-1 Env proteinto a mammal wherein the protein comprises a stably exposed chemokinereceptor binding site.

[0124] In addition, the use of purified nucleic acid to generate animmune response, where the nucleic acid is in a vector (e.g., a plasmidor a virus), or where the nucleic acid comprises naked nucleic acid notassociated with any other nucleic acid, is well-known in the art. Forexample, methods for construction of nucleic acid vaccines are describedin Burger et al. (1991, J. Gen. Virol. 72:359-367), and are well-knownin the art. See also Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York;Ausubel et al., 1997, Current Protocols in Molecular Biology, Green &Wiley, New York.

[0125] Further, cells expressing the HIV-1 Env protein of choice mayalso be used to generate an immune response to an HIV-1 chemokinereceptor binding site.

[0126] The immune response to the Env immunogen is measured by standardimmunological techniques such as ELISA or Western blotting and othersuch techniques well-known in the art or to be developed in the future.A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. See, e.g., Harlowand Lane (1988, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

[0127] The CD4-independent HIV-1 Env protein of the invention may beformulated in a pharmaceutical composition which is suitable foradministration of the protein to a human or veterinary patient. It willbe appreciated that the precise formulation and dosage amounts will varydepending upon any number of factors, including, but not limited to, thetype and severity of the disease to be treated, the route ofadministration, the age and overall health of the individual, the natureof the Env protein, etc. However, the preparation of a pharmaceuticallyacceptable composition having an appropriate pH, isotonicity, stabilityand other characteristics is within the skill of the art. Pharmaceuticalcompositions are described in the art, for example, in Remington'sPharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton,Pa.).

[0128] The amount of the CD4-independent Env administered, whether it isadministered as protein or as nucleic acid or as a cell expressing HIVenv, is sufficient to elicit an immune response to an HIV-1 chemokinereceptor binding site. The pharmaceutical compositions useful forpracticing the invention may be administered to deliver a dose ofbetween about 1 ng/kg and about 100 mg/kg of patient body weight.Suitable amounts of the CD4-independent Env protein for administrationinclude doses which are high enough to have the desired effect withoutconcomitant adverse effects. When the CD4-independent Env is a proteinor peptide, a preferred dosage range is from about 10 to about 1000 μgof protein or peptide per kg of patient body weight. When theCD4-independent Env is administered in the form of DNA encoding the samecontained within a recombinant virus vector, a dosage of between about10² and about 10¹¹ plaque forming units of virus per kg of patient bodyweight may be used. When naked DNA encoding the CD4-independent Env isto be administered as the pharmaceutical composition, a dosage ofbetween about 10 μg to about several mg of DNA per kg of patient bodyweight may be used.

[0129] In the practice of the methods of the invention, a compositioncontaining a CD4-independent Env protein is administered to a patient ina sufficient amount to treat, prevent, or alleviate a HIV-1 infection inthe individual.

[0130] One skilled in the art would appreciate, based on the disclosureprovided herein, that the Env protein/nucleic acid encoding Env may beadministered to a patient to prevent HIV infection by interfering withvirus binding to the appropriate chemokine receptor using the virus'chemokine receptor binding site and, thereby preventing infection.Further, the Env protein/nucleic acid encoding env may also treat oralleviate the condition in a previously infected individual byaugmenting the immune response in the person that could, in turn, bebeneficial as an adjunct to antiretroviral pharmacologic therapy. Thatis, the immunogen may boost the immune response to the virus chemokinereceptor binding site thereby generating antibodies which block therequisite interactions between the virus chemokine receptor binding siteand the target cell chemokine receptor.

[0131] The frequency of administration of a CD4-independent Env proteinto a patient will also vary depending on several factors including, butnot limited to, the type and severity of the viral infection to betreated, the route of administration, the age and overall health of theindividual, the nature of the Env protein, etc. It is contemplated thatthe frequency of administration of the Env protein to the patient mayvary from about once every few months to about once a month, to aboutonce a week, to about once per day, to about several times daily.

[0132] Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in parenteral, oral solid andliquid formulations, ophthalmic, suppository, aerosol, topical or othersimilar formulations. In addition to the appropriate Env protein, ornucleic acid encoding same, these pharmaceutical compositions maycontain pharmaceutically-acceptable carriers and other ingredients knownto enhance and facilitate drug administration. Thus such compositionsmay optionally contain other components, such as adjuvants, e.g.,aqueous suspensions of aluminum and magnesium hydroxides, and/or otherpharmaceutically acceptable carriers, such as saline. Other possibleformulations, such as nanoparticles, liposomes, resealed erythrocytes,and immunologically based systems may also be used to administer theappropriate Env protein or nucleic acid encoding it to a patientaccording to the methods of the invention.

[0133] Preferably, the composition of the invention is administered tothe human by a parenteral or intravenous route.

[0134] An Env protein and/or a nucleic acid encoding Env, may beadministered in conjunction with other compounds which are used to treatHIV infection. Such compounds include, but are not limited to, proteaseinhibitors, reverse transcriptases inhibitors (nucleoside andnon-nucleoside analogs), AZT, interferons, interleukin-2, othercytokines, and the like. The choice of which additional compound toadminister will vary depending upon any number of the same types offactors that govern the selection of dosage and administration frequencyof the Env protein or nucleic acid encoding same. Selection of thesetypes of compounds for use in conjunction with an Env protein forpractice of the method of the invention is well within the skill ofthose in the art.

[0135] The invention also includes a vaccine comprising an immunogenicdose of a CD4-independent HIV-1 Env protein. As discussed previouslyelsewhere herein, generation of an immune response to the viruschemokine receptor binding site should block interaction of this virussite to the host chemokine receptor ligand thereby interfering withand/or inhibiting the requisite virus/host cell interaction needed forHIV infection.

[0136] In addition, the invention includes a method of identifying acompound which affects exposure of a gp120 protein chemokine receptorbinding site. The method comprises contacting a cell with the compoundand comparing the amount of labeled gp120 specifically bound to the cellwith the amount of labeled chemokine bound to an otherwise identicalcell not contacted with the compound. In one embodiment, the gp120 ofinterest was ¹²⁵I-labeled and bound to cells expressing variouschemokine receptors in the presence or absence of soluble CD4. However,the present invention should not be construed to be limited toradioiodination or to any particular gp120 or to expression of onlythese chemokine receptors. Rather, the invention should be construed toencompass a variety of protein labels such that binding of the gp120 ofinterest may be quantitated. Such methods are well-known in the art andinclude, but are not limited to, biotinylation, and ³⁵S-cys and ³⁵S-met.

[0137] The invention also includes a method of identifying asmall-molecule which inhibits binding of a chemokine receptor by anHIV-1 gp120 using its chemokine receptor binding site. The methodcomprises contacting a cell with a small-molecule prior to orcontemporaneous with contacting the cell with labeled gp120 with orwithout preincubation of the gp120 with soluble CD4. Then, the amount oflabel bound to the cell is measured thereby detecting the amount oflabeled gp120 bound to the cell. The amount of bound gp120 bound to acell contacted with the small-molecule is compared to the amount ofgp120 bound to a cell not contacted with the small-molecule. If a loweramount of gp120 is bound to the cell contacted with the small moleculecompared to the amount of gp120 bound to the cell which was notcontacted with the small-molecule, this is an indication that contactingthe cell with the small-molecule inhibits binding of HIV-1 gp120 to achemokine receptor using its chemokine receptor binding site.

[0138] One skilled in the art would appreciate, based on the disclosureprovided herein, that such small-molecules are useful therapeuticsinhibiting HIV-1 infection of cells in that such small-molecules wouldinhibit the requisite HIV-1 gp120/chemokine receptor interactionsnecessary for virus infection of the target cell. Further, the prior artteaches that antibodies and chemokines which specifically bind tochemokine receptors and which block gp120 binding to the chemokinereceptor often also block HIV infection (Lee et al., 1999, J. Biol.Chem., in press; Olson et al., 1999, J. Virol., in press; Wu et al.,1997, J. Exp. Med.). Thus, the small-molecule inhibitors of gp120binding to the chemokine receptor identified using the methods of theinvention are useful inhibitors of HIV infection.

[0139] Further, one skilled in the art, based upon the disclosureprovided herein, would appreciate that a small-molecule inhibitor ofgp120 binding using its chemokine receptor binding site to a chemokinereceptor identified using the methods of the invention is a usefulinhibitor of a chemokine binding to and activation of its receptor. Thatis, the small-molecule inhibitor may be useful for inhibiting thenatural function of chemokine receptors unrelated to the role of thechemokine receptors in HIV infection. Thus, a small-molecule inhibitoridentified herein is a useful therapeutic having potential uses for,among other things, immune system treatments, inflammation, anddevelopment in any non-HIV infected human.

[0140] The invention includes a method of inhibiting HIV-1 gp120binding, using its chemokine receptor binding site, to a chemokinereceptor. The method comprises contacting a the gp120 with asmall-molecule which inhibits binding of gp120 to a chemokine receptorwhere such binding is mediated by the chemokine receptor binding site ofthe virus gp120 protein. The small-molecule is identified as disclosedpreviously elsewhere herein. Contacting the gp120 with thesmall-molecule binding inhibitor inhibits binding of the gp120 with thecell chemokine receptor.

[0141] The invention also includes a method of inhibiting HIV-1infection of a cell. The method comprises contacting a cell with asmall-molecule identified as described previously elsewhere herein. Thesmall-molecule so identified inhibits the binding an HIV-1 gp120 to acell chemokine receptor mediated by the virus gp120's chemokine receptorbinding site. The small-molecule, by interfering with the requisitegp120/chemokine receptor interaction(s), thereby inhibits HIV-1infection of the cell. Indeed, it has been demonstrated previously (Leeet al., 1999, J. Biol. Chem., in press; Olson et al., 1999, J. Virol.,in press; Wu et al., 1997, J. Exp. Med.) antibodies and chemokines thatblock gp120 binding to the chemokine receptor often also block HIVinfection. Thus, the invention includes a method of inhibiting HIV-1infection by interfering with the receptor/ligand interactions requiredfor HIV-1 infection of a target cell using a small-molecule inhibitor ofgp120 binding to the cell chemokine receptor using the gp120 chemokinereceptor binding site.

[0142] The invention also includes a composition comprising aCD4-independent HIV-1 Env and at least one compound used to treat HIVinfection in a pharmaceutically suitable carrier. As described elsewhereherein, the HIV-1 Env may be a HIV-1 Env polypeptide, a nucleic acidencoding HIV-1 Env, and/or a cell expressing HIV-1 env. Further, asdisclosed previously elsewhere herein, the invention should be construedto encompass compounds used to treat HIV infection such as, for examplebut not limited to, protease inhibitors, reverse transcriptaseinhibitor, reverse transcriptase inhibitors (including both nucleosideand non-nucleoside analogs), interferons, AZT, interleukin-2, andcytokines.

[0143] The invention includes a method of treating HIV-1 infection in ahuman. The method comprises administering an immunogenic dose of aCD4-independent HIV-1 Env to an HIV-1 infected human. Administration ofsuch CD4-independent HIV-1 Env induces the production of antibodies tothe stably exposed chemokine receptor binding site of gp120. Thus,administration of the CD4-independent HIV-1 Env causes the production ofpotentially neutralizing antibodies which block the gp120/chemokinereceptor interaction(s) required for HIV-1 infection of the host cell.This is suggested by the fact, disclosed elsewhere herein, that theCD4-independent gp120 is more sensitive to neutralizing antibodies thanotherwise identical CD4-dependent gp120 which does not comprise a stablyexposed chemokine receptor binding site. Further, antibodies that blockEnv-chemokine receptor interactions can neutralize HIV-1 (Wu et al.,1996, Nature 384:179-183; Trkola et al., 1996, Nature 384:184-187).Thus, increased exposure of the chemokine receptor binding site willenhance the production of antibodies to this conserved region whichantibodies inhibit the requisite gp120-chemokine receptor interactions.Therefore, immunizing a human with CD4-independent Env causes theproduction of antibodies to the stably exposed chemokine receptorbinding site which antibodies block requisite Env-chemokine receptorinteractions needed for infection, thereby treating HIV-1 infection inthe human.

[0144] One skilled in the art would appreciate, based upon thedisclosure provided herein, that the immunogenic dose of aCD4-independent HIV-1 Env may be a useful therapeutic to treat and/oralleviate the HIV-1 infection in a human both before and after exposureto the HIV-1 virus. That is, the immunogenic dose may be administeredprior to, during, or after infection of a human by HIV-1. Irrespectiveof when it is administered, the immunogen elicits a response in thehuman to, inter alia, the stably exposed chemokine receptor binding siteof gp120 thereby inducing a response which inhibits the binding of thevirus gp120 to the chemokine receptor. This inhibition is generated inboth previously infected individuals as well as uninfected persons. Inthe individual already infected with HIV-1, the immunogen generates animmune response in addition to any immune response already present inthe individual and thus mediates a reduction in the virus load in thatindividual. Thus, the CD4-independent HIV-1 Env is useful as atherapeutic vaccine in a human already infected by HIV-1 virus.

[0145] As disclosed previously elsewhere herein, one skilled in the artwould appreciate, based on the disclosure provided herein, that theimmunogenic dose of a CD4-independent HIV-1 Env may be administered as aprotein, a nucleic acid (comprising a vector or as naked DNA), and/or acell expressing a nucleic acid encoding a CD4-independent env.

[0146] In another aspect, the method of treating HIV-1 infection in ahuman comprises further administering a compound used to treat HIVinfection. As disclosed previously elsewhere herein, such compoundsinclude, but are not limited to, a protease inhibitors, a reversetranscriptase inhibitor, a reverse transcriptase inhibitor (includingboth nucleoside and non-nucleoside analogs), an interferon, AZT,interleukin-2, and a cytokine. The compound may be administered before,during, or after the administration of the immunogenic dose of aCD4-independent HIV-1 Env.

[0147] One skilled in the art would appreciate, based upon thedisclosure provided herein, that the timing of the compound relative tothe immunogenic dose of a CD4-independent HIV-1 Env would depend uponthe immunization regimen regarding the HIV-1 Env and the particularcompound(s) administered with the Env immunogen, as well as the healthand age of the patient and the severity and stage of the diseaseprocess.

[0148] The HIV-1 Env immunogen(s) and/or compounds which are identifiedusing any of the methods described herein may be formulated andadministered to a mammal for treatment and/or prevention of HIVinfection as now described.

[0149] The invention encompasses the preparation and use ofpharmaceutical compositions comprising a compound useful for treatmentof HIV infection as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, as a combinationof at least one active ingredient (e.g., an immunogenic dose of aCD4-independent HIV-1 Env and a compound used to treat HIV infectionsuch as interleukin-2) in a form suitable for administration to asubject, or the pharmaceutical composition may comprise the activeingredient and one or more pharmaceutically acceptable carriers, one ormore additional ingredients, or some combination of these. The activeingredient may be present in the pharmaceutical composition in the formof a physiologically acceptable ester or salt, such as in combinationwith a physiologically acceptable cation or anion, as is well known inthe art.

[0150] As used herein, the term “pharmaceutically acceptable carrier”means a chemical composition with which the active ingredient may becombined and which, following the combination, can be used to administerthe active ingredient to a subject.

[0151] As used herein, the term “physiologically acceptable” ester orsalt means an ester or salt form of the active ingredient which iscompatible with any other ingredients of the pharmaceutical composition,which is not deleterious to the subject to which the composition is tobe administered.

[0152] The formulations of the pharmaceutical compositions describedherein may be prepared by any method known or hereafter developed in theart of pharmacology. In general, such preparatory methods include thestep of bringing the active ingredient into association with a carrieror one or more other accessory ingredients, and then, if necessary ordesirable, shaping or packaging the product into a desired single- ormulti-dose unit.

[0153] Although the descriptions of pharmaceutical compositions providedherein are principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as non-human primates, cattle, pigs, horses,sheep, cats, and dogs, birds including commercially relevant birds suchas chickens, ducks, geese, and turkeys, fish including farm-raised fishand aquarium fish, and crustaceans such as farm-raised shellfish.

[0154] Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active ingredient,and immunologically-based formulations.

[0155] A pharmaceutical composition of the invention may be prepared,packaged, or sold in bulk, as a single unit dose, or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

[0156] The relative amounts of the active ingredient, thepharmaceutically acceptable carrier, and any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

[0157] In addition to the active ingredient, a pharmaceuticalcomposition of the invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers and AZT, protease inhibitors, reverse transcriptaseinhibitors, interleukin-2, interferons, cytokines, and the like.

[0158] Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

[0159] A formulation of a pharmaceutical composition of the inventionsuitable for oral administration may be prepared, packaged, or sold inthe form of a discrete solid dose unit including, but not limited to, atablet, a hard or soft capsule, a cachet, a troche, or a lozenge, eachcontaining a predetermined amount of the active ingredient. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, or an emulsion.

[0160] As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

[0161] A tablet comprising the active ingredient may, for example, bemade by compressing or molding the active ingredient, optionally withone or more additional ingredients. Compressed tablets may be preparedby compressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycolate. Known surface active agents include,but are not limited to, sodium lauryl sulphate. Known diluents include,but are not limited to, calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

[0162] Tablets may be non-coated or they may be coated using knownmethods to achieve delayed disintegration in the gastrointestinal tractof a subject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

[0163] Hard capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

[0164] Soft gelatin capsules comprising the active ingredient may bemade using a physiologically degradable composition, such as gelatin.Such soft capsules comprise the active ingredient, which may be mixedwith water or an oil medium such as peanut oil, liquid paraffin, orolive oil.

[0165] Liquid formulations of a pharmaceutical composition of theinvention which are suitable for oral administration may be prepared,packaged, and sold either in liquid form or in the form of a dry productintended for reconstitution with water or another suitable vehicle priorto use.

[0166] Liquid suspensions may be prepared using conventional methods toachieve suspension of the active ingredient in an aqueous or oilyvehicle. Aqueous vehicles include, for example, water and isotonicsaline. Oily vehicles include, for example, almond oil, oily esters,ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconutoil, fractionated vegetable oils, and mineral oils such as liquidparaffin. Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

[0167] Liquid solutions of the active ingredient in aqueous or oilysolvents may be prepared in substantially the same manner as liquidsuspensions, the primary difference being that the active ingredient isdissolved, rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

[0168] Powdered and granular formulations of a pharmaceuticalpreparation of the invention may be prepared using known methods. Suchformulations may be administered directly to a subject, used, forexample, to form tablets, to fill capsules, or to prepare an aqueous oroily suspension or solution by addition of an aqueous or oily vehiclethereto. Each of these formulations may further comprise one or more ofdispersing or wetting agent, a suspending agent, and a preservative.Additional excipients, such as fillers and sweetening, flavoring, orcoloring agents, may also be included in these formulations.

[0169] A pharmaceutical composition of the invention may also beprepared, packaged, or sold in the form of oil-in-water emulsion or awater-in-oil emulsion. The oily phase may be a vegetable oil such asolive or arachis oil, a mineral oil such as liquid paraffin, or acombination of these. Such compositions may further comprise one or moreemulsifying agents such as naturally occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soybean orlecithin phosphatide, esters or partial esters derived from combinationsof fatty acids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

[0170] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for rectal administration.Such a composition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

[0171] Suppository formulations may be made by combining the activeingredient with a non-irritating pharmaceutically acceptable excipientwhich is solid at ordinary room temperature (i.e., about 20° C.) andwhich is liquid at the rectal temperature of the subject (i.e., about37° C. in a healthy human). Suitable pharmaceutically acceptableexcipients include, but are not limited to, cocoa butter, polyethyleneglycols, and various glycerides. Suppository formulations may furthercomprise various additional ingredients including, but not limited to,antioxidants and preservatives.

[0172] Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

[0173] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for vaginal administration.Such a composition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

[0174] Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

[0175] Douche preparations or solutions for vaginal irrigation may bemade by combining the active ingredient with a pharmaceuticallyacceptable liquid carrier. As is well known in the art, douchepreparations may be administered using, and may be packaged within, adelivery device adapted to the vaginal anatomy of the subject. Douchepreparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, antibiotics, antifungalagents, and preservatives.

[0176] As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

[0177] Formulations of a pharmaceutical composition suitable forparenteral administration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

[0178] The pharmaceutical compositions may be prepared, packaged, orsold in the form of a sterile injectable aqueous or oily suspension orsolution. This suspension or solution may be formulated according to theknown art, and may comprise, in addition to the active ingredient,additional ingredients such as the dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides. Other parentally-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form, in a liposomal preparation, or as acomponent of a biodegradable polymer systems. Compositions for sustainedrelease or implantation may comprise pharmaceutically acceptablepolymeric or hydrophobic materials such as an emulsion, an ion exchangeresin, a sparingly soluble polymer, or a sparingly soluble salt.

[0179] Formulations suitable for topical administration include, but arenot limited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

[0180] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for pulmonary administrationvia the buccal cavity. Such a formulation may comprise dry particleswhich comprise the active ingredient and which have a diameter in therange from about 0.5 to about 7 nanometers, and preferably from about 1to about 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

[0181] Low boiling propellants generally include liquid propellantshaving a boiling point of below 65° F. at atmospheric pressure.Generally the propellant may constitute 50 to 99.9% (w/w) of thecomposition, and the active ingredient may constitute 0.1 to 20% (w/w)of the composition. The propellant may further comprise additionalingredients such as a liquid non-ionic or solid anionic surfactant or asolid diluent (preferably having a particle size of the same order asparticles comprising the active ingredient).

[0182] Pharmaceutical compositions of the invention formulated forpulmonary delivery may also provide the active ingredient in the form ofdroplets of a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

[0183] The formulations described herein as being useful for pulmonarydelivery are also useful for intranasal delivery of a pharmaceuticalcomposition of the invention.

[0184] Another formulation suitable for intranasal administration is acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 to 500 micrometers. Such a formulation isadministered in the manner in which snuff is taken i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

[0185] Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

[0186] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for buccal administration.Such formulations may, for example, be in the form of tablets orlozenges made using conventional methods, and may, for example, 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

[0187] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for ophthalmicadministration. Such formulations may, for example, be in the form ofeye drops including, for example, a 0.1-1.0% (w/w) solution orsuspension of the active ingredient in an aqueous or oily liquidcarrier. Such drops may further comprise buffering agents, salts, or oneor more other of the additional ingredients described herein. Otherophthalmalmically-administrable formulations which are useful includethose which comprise the active ingredient in microcrystalline form orin a liposomal preparation.

[0188] As used herein, “additional ingredients” include, but are notlimited to, one or more of the following: excipients; surface activeagents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents; sweeteningagents; flavoring agents; coloring agents; preservatives;physiologically degradable compositions such as gelatin; aqueousvehicles and solvents; oily vehicles and solvents; suspending agents;dispersing or wetting agents; emulsifying agents, demulcents; buffers;salts; thickening agents; fillers; emulsifying agents; antioxidants;antibiotics; antifungal agents; stabilizing agents; and pharmaceuticallyacceptable polymeric or hydrophobic materials. Other “additionalingredients” which may be included in the pharmaceutical compositions ofthe invention are known in the art and described, for example inRemington's Pharmaceutical Sciences (1985, Genaro, ed., Mack PublishingCo., Easton, Pa.), which is incorporated herein by reference.

[0189] Typically dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per kilogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the compound will vary fromabout 1 mg to about 10 g per kilogram of body weight of the animal. Morepreferably, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

[0190] The compound may be administered to an animal as frequently asseveral times daily, or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenless frequently, such as once every several months or even once a yearor less. The frequency of the dose will be readily apparent to theskilled artisan and will depend upon any number of factors, such as, butnot limited to, the type and severity of the disease being treated, thetype and age of the animal, etc.

[0191] The compound used to treat HIV infection may be co-administeredwith the immunogenic dose of CD4-independent HIV-1 Env. Alternatively,the compound(s) may be administered an hour, a day, a week, a month, oreven more, in advance of the immunogenic dose(s) of HIV-1 Env, or anypermutation thereof. Further, the compound(s) may be administered anhour, a day, a week, or even more, after the immunogenic dose(s) ofHIV-1 Env, or any permutation thereof. The frequency and administrationregimen will be readily apparent to the skilled artisan and will dependupon any number of factors such as, but not limited to, the type andseverity of the disease being treated, the age and health status of theanimal, the identity of the compound or compounds being administered,the route of administration of the various compounds and HIV-1 Env, andthe like.

[0192] The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

[0193] Definitions

[0194] As used herein, each of the following terms has the meaningassociated with it in this section.

[0195] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0196] As used herein, to “alleviate” a HIV-1 infection means reducingthe severity of the symptoms of the disease or disorder.

[0197] The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies (Harlow et al., 1988, In:Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

[0198] By the term “synthetic antibody” as used herein, is meant anantibody which is generated using recombinant DNA technology, such as,for example, an antibody expressed by a bacteriophage as describedherein. The term should also be construed to mean an antibody which hasbeen generated by the synthesis of a DNA molecule encoding the antibodyand which DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

[0199] By “biological activity,” as the term is used herein, is meantthat the protein has the ability to interact with its associatedprotein(s) and effectuate its normal function(s) within the cell and/orwith respect to HIV-1 infection. In one embodiment, the 8x gp120 retainsits biological activity in that the protein does not require interactionwith CD4 in order to bind to CXCR4 chemokine receptor protein, and tomediate fusion of the virus envelope with the host cell membrane.Further, biological activity as it refers to any form or fragment ofEnv, means that the polypeptide has the ability to bind to a chemokinereceptor protein without the requirement that it also bind to CD4.

[0200] By “chemokine receptor binding site,” as the term is used herein,is meant the portion of the viral gp120 which specifically binds thehuman chemokine receptor protein such as, but not limited to, CXCR4 orCCR5. Thus, a CXCR4 chemokine receptor binding site means a portion ofthe HIV-1 gp120 molecule which specifically binds to CXCR4 chemokinereceptor but which does not substantially bind to another chemokinereceptor such as CCR5. Similarly, a CCR5 chemokine receptor binding sitemeans a portion of the HIV-1 gp120 molecule which specifically binds toCCR5 but which does not significantly bind to any other moleculeincluding another chemokine receptor such as CXCR4 and the like.

[0201] By the term “CD4-independence,” as the term is used herein, ismeant that the HIV-1 strain is capable of infecting cells which do notexpress the CD4 protein and/or its gp120 can bind to a coreceptor in theabsence of CD4-induced conformational change(s). However, theCD4-independent HIV-1 may also infect cells which express CD4 and anappropriate chemokine receptor, although this is not required.

[0202] By the term “chimera,” as used herein, is meant a nucleic acidencoding env comprising a portion of the 8x nucleic acid encoding atleast a portion of env covalently linked to at least one nucleic acidencoding a portion of an env from a different HIV-1 strain.

[0203] By the term “Env clone,” as that term is used herein, is meant anenv nucleic acid encoding an Env protein, gp160, comprising gp120 andgp41. A full-length Env clone encodes a complete Env protein, gp160,while a partial clone includes fragment(s) of a full-length clone thatmay be used to construct smaller portions of the 8x Env that maycomprise mutations that are specific for 8x.

[0204] “Complementary” as used herein refers to the broad concept ofsubunit sequence complementarity between two nucleic acids, e.g., twoDNA molecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

[0205] The use of the terms “nucleic acid encoding” or “nucleic acidcoding” should be construed to include the RNA or DNA sequence whichencodes the desired protein and any necessary 5′ or 3′ untranslatedregions accompanying the actual coding sequence.

[0206] By the terms “encoding” and “coding,” as these terms are usedherein, is meant that the nucleotide sequence of a nucleic acid iscapable of specifying a particular polypeptide of interest. That is, thenucleic acid may be transcribed and/or translated to produce thepolypeptide. Thus, for example, a nucleic acid encoding HIV-1 Env iscapable of being transcribed and/or translated to produce an HIV-1envelope protein.

[0207] As used herein, the term “fragment” as applied to a polypeptide,may ordinarily be at least about seven contiguous amino acids,typically, at least about fifteen contiguous amino acids, moretypically, at least about thirty contiguous amino acids, typically atleast about forty contiguous amino acids, preferably at least aboutfifty amino acids, even more preferably at least about sixty amino acidsand most preferably, the peptide fragment will be greater than aboutsixty contiguous amino acids in length.

[0208] “Homologous” as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., between two nucleicacid molecules, e.g., two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ ATTGCC 5′ and 3′ TATGCG 5′ share 50%homology.

[0209] Further, algorithms may be used to calculate the percent homologybetween two nucleic acids or two proteins of interest and these arewell-known in the art.

[0210] By the term “immunogenic dose,” as the term is used herein, ismeant an amount of protein, whether it is administered as protein or asnucleic acid, which generates a detectable humoral and/or cellularimmune response to the protein compared to the immune response of anotherwise identical mammal to which the protein is not administered. Inone aspect, the dose is administered as Env protein or a fragmentthereof. In another aspect, the dose is administered as a nucleic acid.

[0211] By the term “isolated nucleic acid,” as used herein, is meant anucleic acid sequence, or a fragment thereof, which has been separatedfrom the sequences which flank it in a naturally occurring state, e.g.,a DNA fragment which has been removed from the sequences which arenormally adjacent to the fragment, e.g., the sequences adjacent to thefragment in a genome in which it naturally occurs. The term also appliesto nucleic acids which have been substantially purified from othercomponents which naturally accompany the nucleic acid, e.g., RNA or DNAor proteins, which naturally accompany it in the cell. The termtherefore includes, for example, a recombinant DNA which is incorporatedinto a vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote; or which exists as aseparate molecule (e.g., as a cDNA or a genomic or cDNA fragmentproduced by PCR or restriction enzyme digestion) independent of othersequences. It also includes a recombinant DNA which is part of a hybridgene encoding additional polypeptide sequences.

[0212] By the terms “isolated peptide,” “isolated polypeptide,” or“isolated protein,” as used herein, is meant a peptide or protein whichhas been substantially separated from the components, e.g., DNA, RNA,other proteins and peptides, carbohydrates and lipids, which naturallyaccompany the protein or peptide in the cell. The terms isolated peptideand protein may be construed to include a peptide or protein which isexpressed and/or secreted from a cell comprising an isolated nucleicacid.

[0213] “Mutants,” “derivatives,” and “variants” of the peptides of theinvention (or of the DNA encoding the same) are peptides which may bealtered in one or more amino acids (or in one or more base pairs) suchthat the peptide (or DNA) is not identical to the sequences recitedherein, but has the same property as the peptides disclosed herein, inthat the peptide has the property of binding to a chemokine receptorprotein in a CD4-independent manner.

[0214] As used herein, the term “pharmaceutically-acceptable carrier”means a chemical composition with which an appropriate Env protein, maybe combined and which, following the combination, can be used toadminister the protein to a patient.

[0215] By the term “specifically binds,” as used herein, is meant achemokine receptor binding site which recognizes and binds, for example,CXCR4 polypeptide, but does not substantially recognize or bind othermolecules in a sample. Similarly, a chemokine receptor binding site“specifically binds CXCR4” if the binding site recognizes and bindsCXCR4 in a sample but does not substantially recognize or bind to othermolecules, e.g., CCR5, in a sample. Similarly, a chemokine receptorbinding site may specifically bind CCR5 and, thus, would not bind othermolecules such as CXCR4.

[0216] A swarm refers to an uncloned stock of HIV from infected cells.Such stocks are known to contain many genetically distinct variants of afounder or a parental virus, hence the term “swarm.”

[0217] The term “stably exposed chemokine receptor binding site,” asused herein, means that the gp120 chemokine receptor binding site isavailable to bind to the chemokine receptor protein without the need forgp120 interaction with CD4 which typically, is a prerequisite to gp120binding of the chemokine receptor protein. As demonstrated by the datadisclosed herein, the chemokine receptor binding site of gp120 can existin a stable, exposed configuration which is more sensitive to antibodyneutralization than the otherwise identical CD4-dependent gp120 prior tobinding of CD4. The stably exposed form of the chemokine binding sitecan exist in solution for a period of at least about three months and/orindefinitely.

[0218] As used herein, the term “substantially pure” describes acompound, e.g., a nucleic acid, protein or polypeptide, which has beenseparated from components which naturally accompany it. Typically, acompound is substantially pure when at least about 10%, preferably atleast about 20%, more preferably at least about 50%, still morepreferably at least about 75%, even more preferably at least about 90%,and most preferably at least about 99% of the total material (by volume,by wet or dry weight, or by mole percent or mole fraction) in a sampleis the compound of interest. Purity can be measured by any appropriatemethod, e.g., by column chromatography, gel electrophoresis or HPLCanalysis.

[0219] A compound, e.g., a nucleic acid, a protein or polypeptide isalso “substantially purified” when it is essentially free of naturallyassociated components or when it is separated from the nativecontaminants which accompany it in its natural state. Thus, a“substantially pure” preparation of a nucleic acid, as used herein,refers to a nucleic acid sequence which has been purified from thesequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment in a genome in which it naturally occurs.

[0220] Similarly, a “substantially pure” preparation of a protein or apolypeptide, as used herein, refers to a protein or polypeptide whichhas been purified from components with which it is normally associatedin its naturally occurring state.

[0221] As used herein, to “treat” means reducing the frequency withwhich symptoms of the HIV-1 infection are experienced by a patient.

[0222] By “triggered,” as the term is used herein, it is meant that theHIV-1 Env protein does not require binding to CD4 before gp120 can bindto a chemokine receptor protein such as CXCR4 or CCR5. Preferably, atriggered Env comprises a gp120 that is in a conformation that can bindchemokine receptors in the absence of binding to CD4.

[0223] By the term “vector” as used herein, is meant any plasmid orvirus encoding an exogenous nucleic acid. The term should also beconstrued to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into virions or cells, such as, forexample, polylysine compounds and the like. The vector may be a viralvector which is suitable as a delivery vehicle for delivery of the HIV-1Env protein or nucleic acid encoding the HIV-1 env, to the patient, orthe vector may be a non-viral vector which is suitable for the samepurpose. Examples of viral and non-viral vectors for delivery of DNA tocells and tissues are well known in the art and are described, forexample, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A.94:12744-12746). Examples of viral vectors include, but are not limitedto, a recombinant vaccinia virus, a recombinant adenovirus, arecombinant retrovirus, a recombinant adeno-associated virus, arecombinant avian pox virus, and the like (Cranage et al., 1986, EMBO J.5:3057-3063; International Patent Application No. WO94/17810, publishedAug. 18, 1994; International Patent Application No. WO94/23744,published Oct. 27, 1994). Examples of non-viral vectors include, but arenot limited to, liposomes, polyamine derivatives of DNA, and the like.

[0224] By the term “vaccine,” as the term is used herein, is meant acompound which when administered to a human or veterinary patient,induces a detectable immune response, humoral and/or cellular, to HIV-1or a component(s) thereof.

EXAMPLE 1 Determinants of CD4-independence for an HIV-1 Map OutsideRegions Required for Coreceptor Specificity

[0225] The experiments presented in this example may be summarized asfollows.

[0226] Although infection by HIV typically requires an interactionbetween the viral envelope glycoprotein (Env), CD4, and a chemokinereceptor, CD4-independent isolates of HIV and SIV have been discoveredherein. The present invention discloses the derivation of a variant ofHIV-1/IIIB, termed HIV-1/IIIBx, which exhibits the ability to utilizeCXCR4 in the absence of CD4. This virus infected CD4-negative T and Bcells and fused with murine 3T3 cells expressing human CXCR4 alone.

[0227] A functional HIV-1/IIIBx env clone exhibited several mutations,including the striking loss of 5 glycosylation sites. The data disclosedherein demonstrate the construction of chimeras with CD4-dependent envs.The data disclose that the determinants for CD4-independence map outsidethe V1/V2 and V3 hypervariable loops, which determine chemokine receptorspecificity, and, at least in part, within an area on the gp120 corethat has been implicated in forming a conserved chemokine receptorbinding site. Further, the data disclosed herein demonstrate that whenthe V3 loop of a CCR5-tropic Env was substituted into the HIV-1/IIIBxEnv, the resulting chimera utilized CCR5 but remained CD4-independent.Thus, the data disclosed demonstrate, for the first time, that Envdeterminants for chemokine receptor specificity are distinct from thosethat mediate use of that receptor for cell fusion. These findingsprovide evidence that mutations in HIV-1/IIIBx expose a conservedchemokine receptor binding site that can interact with either CXCR4 orCCR5 in the absence of CD4 and may have important implications fordesigning Envs with exposed chemokine receptor binding sites for vaccinedevelopment.

[0228] The data presented herein disclose the derivation and molecularcharacterization of a variant of HIV-1/IIIB, termed HIV-1/IIIBx, whichacquired the ability to utilize CXCR4 in the absence of CD4. Afunctional HIV-1/IIIBx env clone (8x) was used to construct chimeraswith a closely related but CD4-dependent env, and the determinants forCD4 independence were shown to map in part, to the conserved chemokinereceptor binding site and outside the variable loops. Remarkably, when8x contained the V3 loop of a CCR5-tropic Env, it utilized CCR5 butmaintained CD4 independence. These findings provide evidence that CD4binding likely exposes a domain on the gp120 core that can interact withgenetically divergent chemokine receptors. This work may have importantimplications in designing HIV-1 Env proteins with exposed chemokinereceptor contact sites that could exhibit novel biochemical andimmunogenic properties.

[0229] The Materials and Methods used in the experiments presented inthis example are now described.

[0230] Cells, Viruses and Infectivity Assays

[0231] Hut-78 and SupT1 are immortalized CD4+ T cell lines. BC7 is aCD4-negative line derived from SupT1 (Endres et al., 1996, Cell87:745-756 ). Uncloned HIV-1/IIIB was obtained in chronically infectedHut-78 cells as described in Popovic et al. (1984, Science 224:497-500).Supernatant virus from this infected Hut-78 cell culture was seriallypassaged onto SupT1 cells from which HIV-1/IIIBx was isolated bysubsequent passage onto BC7. The IIIB/Sup virus was derived from earlypassage HIV-1/IIIB in SupT1. NIH-3T3 cells, untransfected and stablytransfected with human CXCR4, were described in (Deng et al.,1996,Nature 381:661-666). Reverse transcriptase (RT) assays were performed onculture supernatants as described in Endres et al. (1997, Science278:1462-1464). For neutralization assays, BC7 cells were preincubatedwith varying concentrations of anti-CXCR4 MAb, or 12G5 (Endres et al.,1997, supra), for 30 minutes at 37° C., the cells were then inoculatedwith HIV-1/IIIBx (10 TCID₅₀) and the cells were monitored for RTactivity.

[0232] PCR, Cloning. Virus Production and Chimera Construction

[0233] Full-length env coding regions were amplified by PCR from genomicDNA of chronically infected cells using the sense primer5′-CGCAACCTATACCAATAGTAGCAA-3′ [SEQ ID NO:1] and the antisense primer5′-CAGTAAGCCATCCAATCACACTAC-3′ [SEQ ID NO:2] in a BioCycler (Ericomp,San Diego, Calif.). The PCR product was TA-cloned into pCDNA3.1(Invitrogen, San Diego, Calif.) and tested in a reporter gene fusionassay. Functional clones of HIV-1/IIIBx (8x) and IIIB/Sup (S10) weresequenced using an automated sequencer. Clones were also subcloned intopSP73 (Promega Corp., Madison, Wis.) that contained the HXBc2 env usingAsp718 and BamH1 (Hoffman et al., 1998, Proc. Natl. Acad. Sci. U.S.A.95:11360-11365).

[0234] Functional env clones were sub-cloned into the 3′ hemigenome ofpNL4-3 (from the EcoRI site) using unique NdeI and BamHI restrictionsites in env, which encompass the mutations in HIV-1/IIIBx and IIIB/Sup.Virus was generated by digesting 20 μg each of the 5′ pNL4-3 Δvprhemigenome (to the EcoRI site) (Gibbs et al., 1994, AIDS Res. Hum.Retroviruses. 10:343-350) and the various 3′ hemigenome constructs withEcoRI, phenol extracting, and coprecipitating before transfection intoBC7 and SupT1 by electroporation. Cells were monitored for syncytiaformation and supernatant virus harvested to generate virus stocks.HIV-1/IIIB virus stocks were frozen at −70° C. in 1 ml aliquots.HIV-1/IIIBx and 8x virus stocks were frozen at −140° C. in 5% sucrose topreserve infectivity. Chimeras between 8x and S10 or HXBc2 (Hoffman etal., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:11360-11365) wereconstructed using a BsaBI site (nt 7673) to isolate changes in SU (i.e.,the gp120 portion of Env which is the surface portion of Env) from TM(i.e., the gp41 portion of Env which is the transmembrane portion ofEnv) and DraIII (nt 6714), StuI (nt 6948), and Bsu36I (nt 7430) toisolate V1-V2, V3, and V4/C4 regions, respectively. Clones containingthe V3 loop of an R5 virus were constructed by subcloning theAsp718-BamHI fragment from a proviral clone of HXB with the V3 loop ofBaL (Hwang et al., 1991, Science 253:71-74) into pSP73-HXBc2 (Hoffman etal., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:11360-11365). A version of8x containing the V3-loop of BaL was made in a similar fashion, byinserting the StuII-Bsu36I fragment of this provirus into pSP73-8x.

[0235] Cell-cell Fusion Assay

[0236] The ability of env genes to mediate cell-cell fusion wasevaluated using a luciferase-based gene reporter assay (Rucker et al.,1997, Methods Enzymol. 288:118-133). Briefly, quail QT6 cells wereco-transfected with plasmids containing HIV envs by CaPO₄ and infectedwith a vaccinia virus expressing T7 RNA polymerase (Alexander et al.,1992, 1992, J. Virol. 66:2934-2942). These cells were mixed with quailQT6 cells transiently expressing human CXCR4 or CCR5 with or withouthuman CD4 and the luciferase gene under the control of the T7 promoter.Fusion was quantified by lysing the cells 7-8 hours after combining thecells and measuring luciferase expression with a luminometer.

[0237] Reverse Transcriptase Assays

[0238] The productive infection of cells was documented by detection ofthe reverse transcriptase (RT) activity in the culture supernatant aspreviously described (Hoxie et al., 1985, Science 229:1400-1402).Briefly, virus from 1 ml of clarified culture supernatant was pelletedat 100,000×g for 30 minutes at 4° C. and the virus was solubilized in100 μl solubilizing buffer (0.15 M Tris pH 8, 0.4 M NaCl, 0.25% TritonX-100, 10% glycerol, 0.5 mM D.T.). Duplicate 20 μl aliquots were mixedwith 85 μl RT cocktail (67.5 mM Tris pH 7.5, 1.3 mM D.T., 1 mM ATP, 13.5mM MgCl₂ containing 0.05 units poly r(A) and 12.5 μCi ³H-dTTP) andincubated for 1 hour at 37° C. The tubes were placed on ice, 225 μg tRNAwas added to each tube, and RNA was precipitated with cold 10% TCA.Precipitated RNA was captured on a glass fiber filter, and the RNA waswashed with TCA and EtOH. The filters were dried and the radioactivitypresent on each filter (in counts per minute, cpm) was determined in ascintillation counter (LKB/Wallac, Turku, Finland).

[0239] Mutagenesis

[0240] Point mutations were engineered into Env constructs in pSP73using the Quickchange™ Site Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.) according to the manufacturer's specifications. Thefollowing primer pairs produced the D368R mutation that ablated CD4binding: D368R-forward, 5′CCTCAGGAGGGGACCCAGAAATTGTAACGC-3′ (SEQ IDNO:5); D368R-reverse, 5′GCGTTACAATTTCTGGGTCCCCTCCTGAGG-3′ (SEQ ID NO:6).Reciprocal exchange of residues at position 431 in 8x and S10 wasaccomplished using two sets of oligonucleotides: (SEQ ID NO:7)8x-G431E-forward 5′-GGCAGGAAGTAGAAAAAGCAATGTATGCC CC-3′ and (SEQ IDNO:8) 8X-G431E-reverse 5′GGGGCATACATTGCTTTTTCTACTTCCTGC C-3′ and (SEQ IDNO:9) S10-E431G-forward 5′-GGCAGGAAGTAGGAAAAGCAATGTATGCC CC-3′ and (SEQID NO:10) S10-E431G-reverse 5′-GGGGCATACATTGCTTTTCAATGTATGCC CC-3′.

[0241] Western Blot

[0242] Virus isolated from the supernatant of an infected cell culturewas pelleted at 100,000×g for 90 minutes at 4° C., and the virus pelletresuspended in lysis buffer (20 mM Tris pH 8.0, 120 mM NaCl, 0.2% sodiumdeoxycholate, 0.5% NP-40, 0.2 mM EGTA, 0.2 mM NaF, 1 μM pepstatin, 5μg/in leupeptin, 5 μg/ml aprotinin) on ice. Equal volumes of lysate and2× sample buffer (50 mM Tris, pH 6.8, 2% SDS, 30% glycerol, 10%β-mercaptoethanol, 0.2% pyronine Y) were boiled for 7 minutes, chilledon ice for 7 minutes, and the samples were then run on a 12% SDS-PAGEgel.

[0243] The proteins were transferred from the gel to nitrocellulose(BioRad Laboratories, Richmond, Calif.) using a Multiphor II semi-dryelectrotransfer apparatus (Pharmacia-LKB Biotechnology Inc., Piscataway,N.J.). The presence of HIV-1 transmembrane proteins was detected on theblot using the D12 mouse monoclonal antibody as described in Earl et al.(1997, J. Virol. 71:2674-2684), followed by biotinylated sheepanti-mouse IG (heavy and light chains) (Jackson ImmunoResearchLaboratories, West Grove, Pa.), streptavidin-conjugated to horse radishperoxidase (streptavidin-HRP, Amersham, Arlington Heights, Ill.) andchemiluminescence substrate (Pierce Chemical Co., Rockford, Ill.).

[0244] The Results of the experiments presented in this example are nowdescribed.

[0245] Derivation of a CD4-independent Variant of HIV-1/IIIB

[0246] A CD4-independent variant of HIV-1/IIIB was derived by serialpassage of an uncloned stock of HIV-1/IIIB in SupT1 and then inoculatingBC7, a CD4-negative line of SupT1 (Endres et al., 1996, Cell87:745-756). In one experiment, approximately 5% of BC7 cells werepositive for viral p24^(gag) by immunofluorescence assay (IFA). Virusfrom this culture was passaged twice onto uninfected BC7 cells and achronically infected line was established. Virus from this line, termedHIV-1/IIIBx, was compared to an earlier passage of HIV-1/IIIB in SupT1cells, designated IIIB/SupT1. As shown in FIG. 1A, only HIV-1/IIIBxcould infect BC7 while both viruses were able to infect SupT1.HIV-1/IIIBx was also able to infect Raji cells, a CD4-negative Blymphoblastoid cell line. Infection of BC7 could be completely inhibitedby the anti-CXCR4 MAb, 12G5 (FIG. 1B), indicating that this infectionwas likely mediated by CXCR4. Moreover, HIV-1/IIIBx-infected BC7 cellsinduced syncytia when cocultured with murine 3T3 fibroblasts that stablyexpressed human CXCR4 while no fusion was induced on untransfected 3T3cells (FIG. 1C). In addition, no fusion was observed whenHIV-1/IIIB-infected HUT-78 cells were cocultured with theCXCR4-expressing 3T3 cells. Together, these data indicate that theHIV-1/IIIBx variant can utilize CXCR4 as a primary receptor in theabsence of CD4 on T and B lymphoid cell lines and murine fibroblasts.

[0247] Cloning and Characterization of a Functional HIV-1/IIIBx env

[0248] A full length env clone of HIV-1/IIIBx (designated 8x) (SEQ IDNO:4) was amplified by PCR from infected BC7 cells, cloned into pSP73,and compared to a prototypic CD4-dependent IIIB clone (HXBc2) in a cellfusion assay. Both 8x and HXBc2 were able to mediate fusion on quail QT6cells expressing both CD4 and CXCR4, but only 8x could fuse with cellsthat expressed CXCR4 alone (FIG. 2). Of note, 8x fusion was enhancedwhen CD4 and CXCR4 were co-expressed, indicating that the 8x Env waslikely still able to interact with CD4. Additionally, the 8x env clonedinto the pNL4-3 provirus generated a replication competent virus thatinfected SupT1 as well as BC7 cells (FIG. 3A), providing further proofthat the 8x Env was able to utilize CXCR4 in the absence of CD4, andthat the 8x clone was representative of the uncloned parental IIIBxvirus.

[0249] The CD4-independence of the 8x was further evaluated byintroducing an Asp to Arg substitution at gp120 amino acid position 368.This Asp is highly conserved among all HIV-1 isolates and is a criticaldeterminant for CD4 binding by forming a salt bridge with Arg-59 in theCDR2 loop of CD4 (Kwong et al., 1998, Nature 393:648-659). Mutations atthis position have been shown to ablate CD4-binding (Olshevsky et al.,1990, J. Virol. 64:5701-5705). As shown in FIG. 2, although a D368R(i.e., Asp to Arg at amino acid 368) mutation abrogated fusion by HXBc2on cells that coexpressed CD4 and CXCR4, this mutation did not affect8x-mediated fusion on target cells expressing only CXCR4. Unlike the 8xclone, fusion of 8x-D368R was not enhanced when CD4 was co-expressedwith CXCR4, confirming that the 8x-D368R Env was unable to interact withCD4. Thus, the 8x Env was not only able utilize CXCR4 in the absence ofCD4, but could tolerate a mutation that destroyed the CD4 binding siteon gp120.

[0250] Sequence Analysis of env Clones

[0251] Sequences of 8x (SEQ ID NO:3) and S10 (SEQ ID NO:12) werecompared to the published sequence of HXBc2 (SEQ ID NO:11) (FIG. 4).While the number of mutations in 8x is large (16 in gp120 and 7 in TM),6 of the mutations in gp120 and 2 in TM have been observed in other envclones derived from HIV-1/IIIB and, thus, are likely not involved in theCD4-independent phenotype. In gp120, 8 of the 11 unique mutations werein the hypervariable loops, V1/V2 (S143G, I165K, G167S, Q170K, andT188P), V3 (R298K, Q310H, and I320V), and V4 (N386K). Three mutationswere in the gp120 core (D62E, N339S, I423V). Interestingly, 5 mutationsin the gp120 resulted in the loss of potential N-linked glycosylationsites, and 4 of these (S143G, T188P, N339S, and N386K) were unique to8x. The 4 8x-specific mutations in the external domain of TM werelocated within the two regions that form coiled coils (T536A, L544S,N651I and K655M). Remarkably, 8x also contained a single nucleotidedeletion in the TM membrane-spanning domain that introduced aframe-shift at position 706 generating a divergent cytoplasmic tail ofonly 30 amino acids. This feature is surprising since HIV-1 viruses withtruncated cytoplasmic tails typically have been attenuated ornon-infectious (Shimizu et al., 1992, Virology 189:534-546; Dubay etal., 1992, J. Virol. 66:6616-6625; Chen et al., 1996, Virology226:260-268). Nonetheless, as noted above, the 8x Env was able togenerate a replication competent virus that could infect SupT1 as wellas BC7 cells (FIG. 3A). Moreover, western blots of viral lysates fromuncloned HIV-1/IIIBx as well as from NL4-3 containing the 8x envdemonstrated a TM of approximately 35 kD compared to 41 kD for parentalHIV-1/IIIB (FIG. 3B).

[0252] Mapping CD4-independence Using Chimeric Env Proteins

[0253] To identify determinants of CD4-independence, a set of reciprocalchimeras was generated between 8x and HXBc2. Unique restriction siteswere chosen to isolate mutations in gp120 from those in TM and to definethe effects of mutations in the V1/V2, V3, and the V4/C4 subdomains(FIG. 5). Chimeras were cloned into pSP73 and were analyzed in fusionassays as described herein on target cells expressing CXCR4 alone orwith CD4. The results for each chimera are expressed as the percentageof fusion activity of the HXBc2 Env fusion activity on target cells thatexpressed both CXCR4 and CD4. All chimeras were functional when CXCR4and CD4 were coexpressed on the target cells, although considerablequantitative differences were detected with fusion activities rangingfrom about 50% to about 500% that of HXBc2 (FIG. 6). Reciprocal chimerasthat exchanged the entire gp120 and TM were CD4-dependent, although anassessment of the 8x gp120 with an HXBc2 TM [8x (gp120)] was somewhatlimited due to the poor overall fusion activity of this Env. Of note,chimeras that contained the 8x TM were more fusogenic than HXBc2 orchimeras that contained an HXBc2 TM, suggesting that determinants in the8x ectodomain or the prematurely truncated cytoplasmic tail werecontributing to the increased fusogenicity of these clones. Nonetheless,these finding suggested that determinants for CD4-independence were notentirely restricted to gp120 or TM.

[0254] Among chimeras that introduced gp120 subdomains of HXBc2 into an8x background, replacement of V1/V2 and V3 loops either individually orin combination failed to eliminate CD4-independence (FIG. 6; seechimeras HX(V1/V2), HX(V3), HX(V1-V3)). This finding was of interestgiven the importance of these loops as determinants of chemokinereceptor specificity (Cho et al., 1998, J. Virol. 72:2509-2515; Choe etal., 1996, Cell 85:1135-1148; Cocchi et al., 1996, Nature Med.2:1244-1247; Hoffman et al., 1998,.Proc. Natl. Acad. Sci. USA95:11360-11365; Ross and Cullen, 1998, Proc. Natl. Acad. Sci. USA95:7682-7686; Speck et al., 1997, J. Virol. 71:7136-7139). For thesechimeras, fusion activity relative to 8x was reduced (80%) on bothCD4-negative and -positive cells, although CD4-dependent fusion wasstill greater than that seen with HXBc2. In contrast, fusion activity ofHX(v4/C4), which contained the HXBc2 V4/C4, was reduced on CD4 negativecells but was unchanged on CD4+ cells. Interestingly, when both the V3and V4/C4 domains of 8x were replaced with those of HXBc2 [HX(V3-C4)],CD4-independent fusion was completely abrogated, while fusion in thepresence of CD4 was unaffected.

[0255] For chimeras in which domains of 8x were placed on an HXBc2background, no single region of gp120 was able to conferCD4-independence to HXBc2, consistent with evidence noted above thatdeterminants in both gp120 and TM are required (FIG. 6). However, anHXBc2 chimera that contained both the V4/C4 and TM domains from 8x[8x(V4-TM)] was highly competent for both CD4-dependent and independentfusion. Collectively these findings with 8x- and HXBc2-based chimerasindicate that determinants in the 8x gp120 V3 and, particularly, theV4/C4 domain contribute to the CD4-independent phenotype of 8x, but onlywhen associated with the 8x TM.

[0256] Evaluation of a CD4-dependent Clone from IIIBx-infected Cells

[0257] A CD4-dependent Env was also derived from IIIBx-infected SupT1cells. This clone, termed S10, was able to mediate fusion on QT6 cellsthat coexpressed CXCR4 and CD4, but was unable to fuse in the absence ofCD4 (FIG. 7). Sequence analysis demonstrated that S10 shared severalmutations with 8x relative to HXBc2 (8 in gp120 and 3 in gp41) (FIG. 4).In addition, S10 contained several unique mutations: in gp120, G431E inC4 and S461N in V5, and in TM, six additional amino acid changes in theecto- and membrane spanning domains, including the loss of predictedN-lined glycosylation sites at positions 611 and 674 (FIG. 4). S10 alsocontained a 55 nt deletion in the TM cytoplasmic tail that, similar to8x, produces a frameshift mutation and a prematurely truncatedcytoplasmic tail. This deletion also disrupts the rev open reading frameby eliminating the nuclear localization signal at the N terminus andintroducing a frame-shift mutation that truncates the protein before theRRE-binding site (Pollard and Malim, 1998, Annu. Rev. Microbiol.52:491-532). Finally, S10 lacked several changes that were present inthe 8x gp120 (S143G, G167S, Q310H, and I423V) and TM (N651I). Eventhough S10 was functional in fusion assays on CD4+/CXCR4+ target cells,this env was unable to generate infectious virus when cloned into NL4-3,likely as a result of its non-functional Rev Protein.

[0258] Because the S10 Env shared several mutations with 8x but wascompletely CD4-dependent, a set of chimeras between 8x and S10 were madeto identify the determinants for this change. As shown in FIG. 7,chimeras that contained the 8x V3 and V4/C4 [8x(V3-C4)] or V4/C4 alone[8x (V4/C4] on an S10 background exhibited some CD4-independence whilethe reciprocal chimeras on an 8x background, [S10(V3-C4)] and[S10(V4/C4)], were completely CD4-dependent. Because the S10 V4/C4domain contained a unique G431E mutation, the possibility that thischange could have a negative effect on CD4-independence was considered.Indeed, when a Gly was restored at this position in S10 (S10-E431G),this clone exhibited a limited degree of CD4-independence onCXCR4-expressing target cells. Moreover, when the G431E mutation wasintroduced into 8x (8x-G431E), this clone remained fusion competent butbecame completely CD4-dependent (FIG. 7). Thus, a charge change withinthe C4 domain was sufficient to abrogate CD4-independence of 8x, andthese data further support the mapping data obtained using the 8x/HXBc2chimeras described above that implicated this region as being criticalto the CD4-independent phenotype.

[0259] CCR5-tropic V3 Loop Alters Chemokine Receptor Specificity but notCD4-independence

[0260] Given the importance of the V3 loop in determining chemokinereceptor specificity and the evidence that determinants for CD4independence were located outside this domain, the extent to whichtropism and CD4-independence of 8x could be dissociated was examined. AnHXB2 gp120 that contained the V3 loop from the macrophage/CCR5-tropicisolate HIV-1/BaL (HXB2-V3BaL) (Hwang et al., 1991, Science 253:71-74)was used to introduce the BaL V3 loop into 8x. The resulting chimera(8x-V3BaL) was compared to 8x, HXBc2 and HXB2-V3BaL in fusion assays ontarget cells that expressed CXCR4 or CCR5, in the presence or absence ofCD4 (FIG. 8). As the data disclosed herein demonstrate, HXBc2 andHXB2-V3BaL exhibited fusion on CXCR4- or CCR5-expressing cells,respectively, and their activity was strictly CD4-dependent. Incontrast, the 8x-V3BaL chimera was both CCR5-tropic and CD4-independent.Thus, determinants for CD4-independence of 8x are functionally distinctfrom those that mediate chemokine receptor tropism.

[0261] The data disclosed herein demonstrate the derivation andcharacterization of a CD4-independent variant of HIV-1/IIIB, termedHIV-1/IIIBx, that could utilize CXCR4 in the absence of CD4. The 8x envclone of IIIBx was able to generate a replication competent,CD4-independent virus when cloned into an HV-1 provirus and couldmediate fusion on CXCR4-expressing quail cells in the absence of CD4.This clone was also fully functional when Arg was substituted for Asp atgp120 position 368, a residue previously shown to be critical to theformation of the DE4 binding site (Kwong et al., 1998, Nature393:648-659; Olshevsky et al., 1990, J. Virol. 64:5701-5705). Sequenceanalysis of 8x revealed 17 mutations that have not been described inother HIV-11/IIIB proviral clones and a remarkable net loss of 5glycosylation sites on gp120. Reciprocal chimeras between 8x and arelated CD4-dependent clone, HXBc2, indicated that the determinants forCD4 independence mapped outside the hypervariable V1/V2 and V3 loops. AnHXBc2 chimera that contained both the V4/C4 and TM domains of 8x wasCD4-independent while chimeras that contained either domain alone wereCD4-dependent. In addition, a CD4-dependent clone from the IIIBx swarm,S10, that contained a unique G431E mutation in the gp120 C4 domainbecame CD4-independent when this mutation was corrected. Introduction ofthe G431 E mutation into 8x rendered this Env completely CD4-dependent,indicating that a charge change at this position was sufficient todisrupt CD4-independent but not CD4-dependent utilization of CXCR4.Collectively, these findings indicate that a chemokine receptor bindingsite exists on the gp120 core, and that mutations in this region can, inassociation with alterations in TM, render an HIV-1 Env fully functionalin the absence of CD4.

[0262] The HIV-1 V3 loop has been shown to be a principal determinantfor chemokine receptor specificity for CCR5 or CXCR4 following CD4binding (Cho et al., J. Virol. 72:2509-2515; Choe et al., 1996, Cell85:1135-1148; Cocchi et al., 1996, Nature Med. 2:1244-1247; Speck etal., 1997, J. Virol. 71:7136-7139; Trkola et al., 1998, J. Virol.72:1876-1885; Wu et al., 1996, Nature 384:179-183). More recently, theV1/V2 region has also been shown, in the context of an appropriate V3,to mediate use of additional chemokine receptors including CCR3, CCR2b,STRL33, and APJ (Hoffman et al., 1998, Proc. Natl. Acad. Sci. USA95:11360-11365; Ross and Cullen, 1998, Proc. Natl. Acad. Sci. USA95:7682-7686) suggesting that cooperative interactions between V1/V2 andV3 are involved in chemokine receptor recognition. These loops are knownto undergo conformational changes following CD4 binding (Jones et al.,1998, J. Biol. Chem. 273:404-409; Moore et al., 1994, J. Virol.68:469-484; Wu et al., 1996, Nature 384:179-183; Wyatt et al., 1992, J.Virol. 66:6997-7004) that may facilitate an interaction with aparticular chemokine receptor (Jones et al., 1998, J. Biol. Chem.273:404-409; Wu et al., 1996, Nature 384:179-183; Wyatt et al., 1992, J.Virol. 66:6997-7004). However, while these findings have suggested thatV3 itself may contain a chemokine receptor binding site, the markedgenetic diversity of V3 loops among CCR5- or CXCR4-tropic virusesindicates either that these loops contain a common structural element orthat other regions on Env also contribute to chemokine receptorutilization. Recently, mutagenesis of a CCR5-tropic HIV-1 gp120 hasidentified a probable CCR5 binding site on Env that is formed by abridging sheet that connects the inner and outer domains of the gp120core. This region is located between the bases of the V1/V2 and V3 loopsand is predicted to be oriented towards the cell membrane following CD4binding (Rizzuto et al., 1998, Science 280:1949-1953). The remarkableconservation of amino acids in this region among CCR5- and CXCR4-tropicEnvs has suggested that this site could represent a generic chemokinereceptor binding domain capable of interacting with multiple chemokinereceptors. These findings are consistent with a model in whichCD4-induces movement of the V1/V2 and V3 loops, which facilitates aninitial interaction with a specific chemokine receptor and exposes thisconserved binding site that is then required for fusion to occur(Rizzuto et al., 1998, Science 280:1949-1953; Wyatt and Sodroski, 1998,Science 280:1884-1888).

[0263] Because determinants for CD4-independence of the 8x clone mappedoutside regions required for chemokine receptor specificity, thepossibility that a different V3 might change the chemokine receptortropism of 8x without affecting its CD4-independence was investigated.Remarkably, the data disclosed herein demonstrate that when the V3 loopfrom a CCR5-tropic Env (HIV-1BaL) was inserted into 8x, the resultingchimera was able to mediate CD4-independent fusion on CCR5-expressingcells. No fusion on CXCR4-expressing cells with or without CD4 wasobserved for this chimera. In contrast, a chimera containing theHIV-1/BaL V3 loop on an HxBc2 background utilized CCR5 but wascompletely CD4-dependent. These data clearly indicate that chemokinereceptor specificity and the utilization of that receptor for fusion aremediated by distinct regions of gp120. Moreover, the data disclosedherein also provide direct evidence that, although specificitydeterminants on V3 are still required, a region on the gp120 core thatis rendered functional on CD4-independent viruses is able to mediatefusion using genetically divergent chemokine receptors.

[0264] The bridging sheet on gp120 noted above is made up largely ofamino acids from the C4 domain and the V1IV2 stem (Rizzuto et al., 1998,Science 280:1949-1953). This region has also been shown to contribute tothe formation of gp120 epitopes that are induced by CD4 binding (Kwonget al., 1998, Nature 393:648-659; Thali et al., 1993, J. Virol.67:3978-3988). Interestingly, the two mutations in the 8x V4/C4 domain(N386K and 1423V) and a third mutation near the base of the V3 loop(R298K) map to positions that immediately flank this area (FIG. 9A). Asthe data disclosed herein demonstrate, an 8x chimera that included thecorresponding V3 and V4/C4 domains from HXBc2 and that lacked thesemutations was highly competent for fusion but was completelyCD4-dependent (FIG. 6). Without wishing to be bound by theory, theremarkable proximity of R298K, N386K, and I423V to the putativechemokine receptor binding domain strongly suggests that these mutationsexpose this site and/or help to present it to the chemokine receptorduring viral attachment. Further, data disclosed elsewhere herein(Example 2, infra) demonstrate that recombinant 8x gp120 is able to bindto CXCR4-expressing cells independently of CD4 and that CD4-inducedepitopes that are partially contained within the gp120 chemokinereceptor domain are stably exposed in the absence of CD4 binding. Inaddition, the G431E mutation in C4, which was sufficient to abrogateCD4-independence on S10 and 8x, is shown by the crystal structure of thegp120 core to be juxtaposed to residues at the base of the V1/V2 stemthat contribute to the chemokine receptor binding site (FIG. 9B).Without wishing to be bound by theory, the acquisition of a negativecharge at this residue could alter the orientation of the V1/V2 loopsand/or affect the conformation of the chemokine receptor binding site ongp120. Regardless of the mechanism, it is apparent that mutations in oraround this chemokine receptor binding site can impact positively ornegatively on the ability of the 8x Env to function without CD4 and isconsistent with the view that CD4 binding improves the overallefficiency and/or avidity of chemokine receptor utilization.

[0265] While the V4/C4 domain is clearly involved with CD4-independenceof IIBx, it is apparent that other regions of the Env also contribute tothis phenotype. A chimera that contained the 8x V4/C4 on an HXBc2background was only CD4-independent when it also contained the 8x TM. Aprevious study by Reeves et al. (1996, J. Virol. 71:1453-1465) of theCD4-independent HIV-2/ROD-B demonstrated that mutations in both gp120and in TM were the minimal requirements for this phenotype (i.e., a Leuto Phe mutation just proximal to the analogous V4 loop of HIV-1, and twomutations in the first heptad repeat of the TM ectodomain). Although theunderlying mechanism for this effect is unclear, regions of the HIV-1 TMhave been implicated in a number of cooperative interactions with thegp120 that could affect its binding to CD4 and/o to chemokine receptors(Cao et al., 1993, J. Virol. 67:2747-2755; Chan et al., 1997, Cell89:263-273; Matthews et al., 1994, Immunol. Rev. 140:93-104). Of note,the gp120 chemokine receptor binding site described above is locatednear the predicted trimer axis of the assembled Env oligomer whereinteractions with TM are likely to occur (Haigwood et al., 1992, J. Med.Primatol. 21:82-90). The data obtained in recent experiments demonstratethat and HXBc2 chimera containing only the 8x V4/C4 and the 8xframeshift mutation in TM was able to mediate CD4-independent fusion toa level approximately 10% that of 8x. Whether this small butreproducible effect is due to an increase in the surface expression ofEnv on transfected cells (LaBranche et al., 1995, J. Virol.69:5217-5227; Mulligan et al., 1992, J. Virol. 66:3971-3975) or whetherthe effect is due to structural alterations in the TM ectodomain (Ritteret al., 1993, Virology 197:255-264; Spies et al., 1994, J. Virol.68:585-591), and/or gp120 (Cao et al., 1993, J. Virol. 67:2747-2755;Chan et al., 1997, Cell 89:263-273; Matthews et al., 1994, Immunol. Rev.140:93-104) remains to be determined.

[0266] Further, 8x contains mutations that are predicted to eliminate 5glycosylation sites in gp120, including N386K as noted previouslyelsewhere herein, which mutation lies adjacent to the putative chemokinereceptor binding site. Carbohydrates have recently been implicated inmodifying the immunogenicity of SIV gp120 and in masking neutralizationepitopes (Reitter et al., 1998, Nature Med. 4:679-684). Without wishingto be bound by theory, it is possible that the loss of one or moreglycosylation sites could also be involved in exposing the chemokinereceptor binding site.

[0267] Although the data disclosed herein have implicated mutations inthe IIIBx V4/C4 and TM as determinants for CD4-independence, it shouldbe noted that mutations in different regions of gp120 have beenassociated with CD4-independence for other HIV-1 isolates. ACD4-independent variant of HIV-1 /NDK has been described that couldinfect HeLa cells using CXCR4 by virtue of a combination of mutations inthe C2, C3, and V3 domains (Dumonceaux et al., 1998, J. Virol.72:512-519). Recent findings by Sodroski et al., have demonstrated thatdeterminants for a CD4-independent, CCR5 tropic variant of HIV-1/ADAmapped to point mutations in the distal region of the V1/V2 stem.Despite these genetic differences, CD4-independent viruses could have asimilar structural basis for this phenotype. In this regard, at leastsome of the changes in CD4-independent HIV-1/NDK and HIV-1/ADA aresimilar to IIIBx, being located near the gp120 bridging sheet where theycould affect the presentation of this region to a chemokine receptor.

[0268] HIV has evolved strategies that enable viral replication tocontinue in spite of a vigorous host immune response (Wei et al., 1995,Nature 373:117-122; Perelson et al., 1996, Science 271:1582-1586).Neutralizing antibodies typically arise late in the course of infection,if at all, and are frequently directed at type-specific rather thangroup-specific determinants on gp120 (Wyatt and Sodroski, 1998, Science280:1884-1888; Moore and Ho, 1995, AIDS 9:S117-S136). The deducedcrystal structure of the gp120 core has suggested that the CD4 bindingdomain and the chemokine receptor binding site are poorly accessibleand/or are concealed within the Env oligomer (Wyatt and Sodroski, 1998,Science 280:1884-1888; Rizzuto et al., 1998, Science 280:1949-1953). Incontrast, the exposed surfaces of gp120 contain hypervariable domainsand carbohydrates that may serve as immunologic decoys for the humoralimmune response (Stamatatos and Cheng-Mayer, 1998, J. Virol.72:7840-7845; Reitter et al., 1998, Nature Med. 4:679-684; Cao et al.,1997, J. Virol. 71:9808-9812). Approaches to expose these conserved andfunctionally critical domains may enable qualitatively different andperhaps more efficacious immune responses to be generated. Recentstudies by LaCasse et al. (1999, Science 283:357-362), have demonstratedthat a fusion-activated form of Env in which conserved neutralizationepitopes on gp120 and gp41 were apparently stabilized was able togenerate a potent and broadly cross-neutralizing antibody response inmice. In this regard, CD4-independent Envs that are derived or designedmay provide a means to present these domains in a biologically relevantcontext. Data disclosed elsewhere herein demonstrate that the 8x gp120exhibits a number of novel immunological and biochemical propertiesincluding the increased exposure of CD4-induced epitopes (see Example 2)and the ability to bind to CXCR4 in the absence of CD4. Future studiesof HIV-1/IIIBx and additional CD4-independent isolates should providepowerful tools to probe the structure and function of the viral envelopeglycoprotein and lead to the rational design of gp120 molecules withaltered immunogenic properties as therapeutic modalities.

EXAMPLE 2 Stable Exposure of the Coreceptor Binding Site in aCD4-independent HIV-1 Envelope Protein

[0269] The experiments presented in this example may be summarized asfollows.

[0270] The data presented previously in Example 1, disclose aCD4-independent HIV-1 virus, HIV-1/IIIBx, that interacts directly withthe chemokine receptor CXCR4 to infect cells in the absence of CD4. Thedata presented herein disclose the underlying mechanism of theCD4-independence by using a novel cloned Env from the HIV-1/IIIBx swarmnamed 8x previously disclosed in Example 1. The 8x Env clone was used toproduce soluble gp120. The data disclosed herein demonstrate that 8xgp120 bound directly to cells expressing only CXCR4 while binding ofIIIB gp120 also required soluble CD4. Further, using an opticalbiosensor, the data disclosed herein demonstrate that CD4-induced (CD4i)epitopes recognized by monoclonal antibodies (MAbs) 17b and 48d weremore exposed on 8x than on IIIB gp120. The ability of 8x gp120 to binddirectly to CXCR4 and to react with MAbs 17b and 48d in the absence ofCD4 indicates that 8x gp120 exists in a partially triggered but stablestate in which the conserved coreceptor binding site in gp120, whichoverlaps with the 17b epitope, is exposed.

[0271] Substitution of the CXCR4-specific V3-loop of 8x with the V3-loopfrom the CCR5 tropic HIV-1/BaL strain resulted in an Env clone thatmediated CD4-independent, CCR5-dependent virus infection. Therefore, thesubstitution of the V3-loop produced a gp120 chimera (8x-BaL) that boundto CCR5 in the absence of CD4. Thus, the data disclosed hereindemonstrate that in a partially triggered Env protein, the V3-loop canalter the specificity of coreceptor use, but does not alter CD4independence. Moreover, the data disclosed herein indicate that CD4independence and chemokine coreceptor binding are dissociable. Further,HIV-1/IIIBx was far more sensitive to neutralization by HIV-positivehuman sera, a variety of anti-IIIB gp120 rabbit antisera, and CD4i MAbsthan was the CD4-dependent IIIB strain. The increased sensitivity ofHIV-1/IIIBx virus to neutralization by antibodies and the stableexposure of a highly conserved region of gp120 suggest novel strategiesfor the development of antibodies and small molecule inhibitors to thisfunctionally important domain.

[0272] The Materials and Methods used in the experiments presented inthis Example are now described.

[0273] Plasmids

[0274] Human CCR5, CXCR4, and CD4 were expressed using the pCDNA3 vector(Invitrogen, San Diego, Calif.). The luciferase gene was expressed undercontrol of the T7 promoter in the pGEM2 vector (Promega Corp., Madison,Wis.). The Envs from the HXBc2 clone of IIIB and 8x were both expressedin the pSP73 vector (Promega Corp., Madison, Wis.). To generate Envconstructs containing the V3 loop of the R5 HIV-1 strain BaL, theKpnI-BamHI fragment in env from the full-length proviral clonepIIIB-V3BaL was cloned into pSP73-IIIB. To produce a version of 8xcontaining the V3 loop of BaL, the StuI-Bsu361 env fragment ofpIIIB-V3BaL was cloned into pSP73-8x. Stop codons were inserted intoeach env plasmid at the gp120/gp41 junction using the Quickchange™Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) to makeconstructs for gp120 production. The identity of all mutants and cloneswas confirmed by DNA sequencing.

[0275] Cell-Cell Fusion Assay

[0276] This assay has been described in more detail elsewhere. Briefly,effector QT6 cells in T25 flasks were infected with recombinant vacciniavirus expressing T7 polymerase (vTF1.1) and transfected with 30 μg ofenv constructs wherein expression was driven by the T7 promoter. TargetQT6 cells were plated in 24-well plates and each well of cells wastransfected with 0.5 μg CD4 and 1.0 μg coreceptor plasmids under thecontrol of the CMV promoter, and 1.5 μg of the luciferase reporterplasmid under the control of the T7 promoter. Following overnightexpression, the effector cells were added to target cells and luciferaseactivity was quantified in cell lysates 7.5 hours after mixing.

[0277] Protein Production and Purification

[0278] 293T cells were infected in T225 flasks with vTF1.1 and the cellswere then transfected with 200 μg of gp120 plasmid. Four hourspost-transfection, the cells were washed with phosphate buffered saline(PBS) and placed in serum-free media for 24 hours. Media was collected,clarified by centrifugation and 0.2 μM filtration prior to addition of0.1% TX-100. Protein in the supernatant was bound to a Galanthis Navaliscolumn (Vector Laboratories, Burlingame, Calif.), washed withmethyl-α-D-mannopyranoside (MES) buffer (20 mM MES, pH 7.0, 0.13 M NaCl,10 mM CaCl₂) and eluted in MES buffer containing 0.5M α-methylmannoside. The eluate was subjected to additional purification, washing,and concentration using an Amicon ultrafiltration system with a 50 kDprotein molecular weight cutoff. HPLC analysis determined that Envprepared in this fashion was highly pure, and accurate proteinconcentrations were determined by amino acid analysis and BCA assay.

[0279] Cell-Surface Binding Assay

[0280] Binding of gp120 to coreceptors was determined as described byDoranz et al. (1999, J. Virol., 73:2752-2761). Briefly, approximately 5μg of each gp120 was iodinated using Iodogen (Pierce) to specificactivities of 12.3 μCi/μg, 7.15 μCi/μg, 47.3 μCi/μg, and 22.0 μCi/μg forIIIB, 8x, IIIB-V3BaL, and 8x-V3BaL, respectively. One-hundred thousandcounts per minute (CPM) was added to about 0.5 to about 1.0×10⁶293Tcells which had been transfected the previous day with 14 μg DNA in atotal volume of 100 μl binding buffer (50 mM Hepes pH 7.4, 5 mM MgCl₂, 1mM CaCl₂, 5% BSA). Soluble CD4 (sCD4) was added at 100 nM whenindicated. The cells were incubated for 1 hour at 25° C.

[0281] Unbound radioactivity was removed by filtering the cells throughWhatman GF/C filters presoaked in 0.3% polyethylenimine, and washingtwice with 4 ml wash buffer (50 mM Hepes pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂,500 mM NaCl). The amount of radioactivity which bound nonspecifically tothe filters in the absence of cells was subtracted from all data points.

[0282] Biosensor Experiments

[0283] All experiments were performed using a BIACORE 2000 (Upsala,Sweden) optical biosensor at 25° C. Approximately 200 RU of sCD4 and thefull length human MAbs 17b and 48d were attached by amine coupling to aresearch grade CM5 chip. A naked sensor surface without antibody or sCD4served as a negative control for each binding interaction. Env which hadbeen serially diluted was run across each sensor surface at 6 differentconcentrations in a running buffer of PBS+0.005% Tween-20 (11 nM to 585nM for gp120, 5 nM to 91 nM for gp120+sCD4). Soluble CD4 was added at an8-fold molar excess to Env at least 30 minutes prior to measuringbinding, and completely eliminated binding of Env to CD4 attached to thesensor surface. Binding and dissociation were measured for 300 secondseach at a flow rate of 30 μl/minute which gave a flow-independentbinding on-rate. The sensor surface was regenerated between each bindingreaction by using 2 washes of 10 mM HCI for 15 seconds at 100 μl/minute,which was found to return the signal completely to baseline withoutdecreasing the binding capacity of the immobilized surface. Each bindingcurve was corrected for nonspecific binding by subtraction of the signalobtained from the negative control flow cell. Kinetic constants forassociation and dissociation were derived from linear transformations ofthe exported binding data of at least 5 concentrations of analyte. Thekinetic parameters obtained were compared to those estimated by fittingthe data to the simple 1:1 Langmuir interaction model using the BIAEvaluation 3.1 software.

[0284] Neutralization Assays

[0285] Neutralization of virus by antisera or MAbs was performed using amodification of the previously described MAGI assay (Chakerian et al.,1997, J. Virol. 71:3932-3939) or luciferase reporter virus systemdescribed by Connor et al. (1995, Virology 206:935-944). Briefly,1.25×10⁵ MAGI cells were plated in a 48-well plate, and the cells wereallowed to adhere. The cells were infected with virus that had beenpre-incubated with serial dilutions of antibody for 1 hour at 37° C. Theamount of virus used was the amount previously determined with the MAGIassay to contain 400-800 infectious units. Twenty-four hours afterinfection, the DP178 inhibitory peptide was added at a finalconcentration of 5 μg/ml to prevent the formation of syncytia. The cellcultures were incubated another 48 hours, fixed and the cells werestained with X-gal. Blue nuclei were quantified using an AlphaImager2000 (AlphaInnotech Corporation, San Leandro, Calif.). For luciferasereporter virus infections, equal amounts of virus, as judged by relativelight units (RLU), were also incubated with serial dilutions of antibodyfor 1 hour at 37° C. Virus was added to GHOST-CCR5 cells in 96-wellplates and cell lysates were measured for luciferase activity 2 dayspost-infection.

[0286] The Results of the experiments presented in this example are nowdescribed.

[0287] Direct Binding of 8x gp120 to CXCR4

[0288] Binding of CD4 to HIV-1 Env induces conformational changesrequired for subsequent Env-coreceptor interactions. These changes arelikely to include increased exposure of an exceptionally well-conserveddomain in gp120 that has been implicated in coreceptor binding (FIG.12). Many SIV and HIV-2 strains can short-circuit this normal entryprocess by utilizing coreceptors for virus entry in a CD4-independentmanner, although their efficiency is typically enhanced when CD4 ispresent. The data presented previously in Example 1 disclose productionof a CD4-independent HIV-1 strain through repeated passaging of HIV-1IIIB on CD4-negative, CXCR4-positive cells (Example 1, supra). Theresulting virus strain (HIV-1/IIIBx) can utilize CXCR4 in the absence ofCD4 to infect a wide variety of cell types (Example 1, supra). An Envclone derived from cells chronically infected with HIV-1/IIIBx, termed8x, maintains this phenotype. Thus, cells expressing 8x Env mediatedfusion with CD4-negative, CXCR4-positive cells. In the presence of CD4,fusion efficiency was enhanced approximately 3-fold (FIGS. 10A and 10B).Fusion mediated by 8x is strictly dependent upon CXCR4, and is notobserved when other coreceptors are expressed (FIGS. 10A and 10B). SinceCD4 is known to induce conformational changes in Env that enable it tointeract with coreceptors and since the 8x Env can mediateCXCR4-dependent fusion in the absence of CD4, experiments were performedto determine whether the 8x Env protein exists in a partially triggeredstate, thereby enabling it to bind CXCR4 in a CD4-independent manner.

[0289] To evaluate conformational differences in the 8x gp120, theability of purified 8x gp120 to bind directly to CXCR4 was examined. Inthis regard, a cell surface binding assay as described by Doranz et al.(1999, J. Virol., 73:2752-2761), and set forth herein, was used in whichradioiodinated gp120, with or without prior incubation with soluble CD4(sCD4), was added to cells expressing the desired coreceptor. The cellswere washed and the amount of bound radiolabeled gp120 was measured. Thedata disclosed herein demonstrate that IIIB gp120 complexed with sCD4bound to cells expressing CXCR4 but not to cells expressing othercoreceptors (FIG. 11). In contrast, the 8x gp120 bound toCXCR4-expressing cells equally well in the presence or absence of sCD4(FIG. 11). Binding to cells expressing vector alone or CCR5 was notobserved. These results demonstrate that unlike the parental IIIB Env,the 8x gp120 exists in a stable conformation that enables it to interactdirectly with CXCR4 in the absence of CD4.

[0290] Exposure of the Coreceptor Binding Site

[0291] Without wishing to be bound by theory, the ability of 8x gp120 tobind to CXCR4 may be the result of increased exposure of sites on gp120that interact with CXCR4. To determine whether there was increasedexposure of these sites, the abilities of IIIB and 8x gp120 to interactwith the CD4i MAbs 17b and 48d were examined. A Fab fragment of 17b wasco-crystallized with gp120 and sCD4 (Kwong et al., 1998, Nature393:648-659), and the data disclosed demonstrate that many of thecontact residues involved in 17b-gp120 interactions are also importantfor coreceptor binding (FIG. 12). Thus, 17b can be used as animmunological surrogate to measure exposure of the conserved, coreceptorbinding site defined by Rizzuto et al. (1998, Science 280:1949-1953).

[0292] Binding of IIIB and 8x gp120, with or without prior incubationwith sCD4, to the CD4i MAbs 17b and 48d was measured using an opticalbiosensor assay method as described elsewhere herein (see materials andmethods, Example 2). This approach makes it possible to measureprotein-protein interactions in real time, and can be used to derive on-and off-rates as well as affinity constants. The desired MAb wascovalently coupled to the sensor surface after which gp120 or gp120-sCD4complexes were applied. A typical sensorgram is shown in FIG. 13. Asexpected, the rate at which HIV-1/IIIB gp120 bound to MAb 17b wasmarkedly increased by prior incubation of Env with sCD4. Once bound to17b, both IIIB gp120 and gp120-sCD4 complexes exhibited negligibleoff-rates. In contrast to IIIB gp120, 8x gp120 bound efficiently to 17bwithout sCD4, exhibiting an on-rate that was an order of magnitudegreater than that of IIIB gp120. Addition of sCD4 enhanced this on-rate2-fold. When complexed with sCD4, both the 8x and IIIB gp120s exhibitedidentical on-rates (Table 1). Interestingly, once bound to 17b, the 8xgp120 exhibited a greater off-rate than did IIIB gp120. This effect maybe due to the Ile to Val mutation in the 8x gp120 at amino acid position423 (FIG. 12), a residue previously shown to be a contact site for 17b(Kwong et al., supra). TABLE 1 ka (1/Ms) kd (1/s) Kd (nM) Binding to 17b8x 1 × 10⁵ 2 × 10⁻³ 15 8x/CD4 2 × 10⁵ 9 × 10⁻⁴ 4.5 HXB 8 × 10³ 2 × 10⁻⁵2.5 HXB/CD4 3 × 10⁵ 5 × 10⁻⁵ 0.2 Binding to 48d 8x 2 × 10⁵ 1 × 10⁻³ 6.08x/CD4 3 × 10⁵ 6 × 10⁻⁴ 2.0 HXB 1 × 10⁴ 5 × 10⁻⁵ 5.0 HIXB/CD4 5 × 10⁵ 3× 10⁻⁵ 0.1

[0293] The data disclosed in Table 1 demonstrate the apparent kineticand equilibrium constants derived from binding of gp120 to CD4iantibodies in biosensor experiments performed as described previouslyelsewhere herein. Briefly, the CD4i antibodies 17b and 48d were attachedto the biosensor surface, and both binding and dissociation of serialdilutions of 8x and HXB gp120 were measured. Binding of serial dilutionsof both Envs which had been premixed with a saturating amount of sCD4was also determined. All bindings were performed at 25° C., and a samplesensorgram is shown in FIG. 13. The best fitted values for the slopes ofthe linearized plots of the data (r²≧0.98) are reported. The parametersestimated by fitting the simple 1:1 Langmuir interaction model globallywere within 15% of the reported values. Values in italics representdissociation rates that were so slow that they were at the limits ofdetection of the biosensor, making the affinity constants derived fromthese values less accurate.

[0294] Analysis of a different CD4i MAb, 48d, yielded results that weresimilar to 17b (Table 1). Finally, IIIB and 8x gp120 moleculesinteracted with CD4 attached to the sensor surface in an identicalfashion. Thus, the mutations in 8x that render it CD4-independent didnot affect CD4 binding to an appreciable degree, but did result ingreater exposure of the 17b epitope, which overlaps with the conservedcoreceptor binding site.

[0295] Dissociation of Coreceptor Choice and CD4-independence

[0296] Both the conserved coreceptor binding site as well as the V1/V2and V3 loops of gp120 play important roles in Env-coreceptorinteractions. Available evidence indicates that the V3 loop and, to alesser extent, the V1/V2 region govern the number and types ofcoreceptors used by a given Env (Hoffman et al., 1998, Proc. Natl. Acad.Sci. U.S.A. 95:11360-11365; Ross and Cullen, 1998, Proc. Natl. Acad.Sci. U.S.A. 95:7682-7686; Speck et al., 1997, J. Virol. 71:7136-7139;Cocchi et al., 1996, Nature Med. 2:1244-1247; Cho et al., 1998, J.Virol. 72:2509-2515; Choe et al., 1996, Cell 85:1135-1148). In contrast,mutations in the conserved coreceptor binding site can affectEnv-coreceptor binding (Rizzuto et al., 1998, Science 280:1949-1953),but it is not clear if this region also plays a role in coreceptorspecificity. The ability of 8x gp120 to interact directly with CXCR4provided an opportunity to determine if coreceptor choice and changes inEnv that expose the coreceptor binding site are dissociable. Previousstudies demonstrated that the introduction of an R5 V3-loop (from HIV-1BaL) into an HIV-1 IIIB background resulted in an Env protein (IIIB-BaL)that used CCR5, but not CXCR4, for virus infection (Ross and Cullen,1998, Proc. Natl. Acad. Sci. U.S.A. 95:7682-7686; Ross et al., 1997, J.Virol. 72:1918-1924). The data disclosed previously herein in Example 1demonstrate, using a cell-cell fusion assay, that substituting the V3loop of the 8x Env with that from an R5 Env (BaL) produced a protein(8x-BaL) that was able to mediate fusion with CCR5-positive,CD4-negative cells. To determine if 8x-BaL could also mediateCD4-independent virus infection, luciferase reporter viruses asdescribed in Connor et al. (1995, Virology 206:935-944) were generatedbearing either IIIB-V3BaL or 8x-V3BaL Env proteins. Virions bearing8x-V3BaL Env mediated CD4-independent, CCR5 -dependent virus infection(FIG. 10B). As was observed with 8x Env in fusion assays, the presenceof CD4 increased the efficiency of virus entry. Neither IIIB-BaL or8x-BaL used CXCR4 in the presence or absence of CD4. Also, IIIB-BAL and8x-BaL gp120 molecules were produced, and data disclosed hereindemonstrate that IIIB-BaL gp120 bound to CCR5 in a CD4-dependent manner,while 8x-BaL bound to CCR5 independently of CD4. Neither protein boundto CXCR4 under any conditions examined. Thus, changes in the V3-loopaffected which coreceptor was used, but did not impact CD4-independence.

Neutralization of HIV-1/IIIBx

[0297] Antibodies that block Env-coreceptor interactions can neutralizeHIV-1 (Wu et al., 1996, Nature 384:179-183; Trkola et al., 1996, Nature384:184-187). The exposed nature of the coreceptor binding site inHIV-1/IIIBx gp120 might therefore be expected to make this virus moresensitive to antibody mediated neutralization. Several SIV and HIV-1strains with modifications in the V1/V2 region have been shown to beneutralization sensitive, presumably because of increased exposure ofconserved determinants (Stamatatos and Cheng-Mayer, 1998, J. Virol.72:7840-7845; Reitter et al., 1998, Nature Med. 4:679-684). Therefore,the relative sensitivities of H-1/IIIB and HIV-1/IIIBx to neutralizationby HIV-positive human sera, and to sera from rabbits immunized witheither IIIB or 8x gp120, were compared and the results are set forth inTable 2. TABLE 2 HIV-1 IIIB HIV-I IIIBx Rabbit Immunogen 50% 90% 50% 90%1169 IIIB 1,934 132 3,081 287 1170 IIIB 1,896 109 93,756 5,616 11718x >10,240 1,005 >163,840 13,748 1172 8x 1,552 94 64,295 4,426 Humansera ZT02575 1,616 46 20,894 4,195 Human sera JT2140 155 11 892 187IIIB- BaL 8x- BaL MAb 14 ng/ml 90 ng/ml 2 ng/ml 15 ng/ml 17b MAb >5,000ng/ml >5,000 ng/ml 3 ng/ml >200 ng/ml* 48d MAb 95 ng/ml 375 ng/ml 45ng/ml 610 ng/ml 50.1

[0298] The data disclosed in Table 2 were obtained by infecting sMAGIcells with equivalent amounts of HIV-1/IIIB and HIV-1/IIIBx. Infectionwas determined 72 hours after infection as previously described byChackerian et al. (1997, J. Virol. 71:3932-3939). For MAbs 17b and 48d,equivalent amounts of luciferase reporter viruses bearing the IIIB-BaLor 8x-BaL Envs were used to infect GHOST-CCR5 cells which were lysed 48hours post-infection. For all infections, virus and serial dilutions ofMAbs, human sera, and rabbit sera were mixed for 1 hour prior toaddition to the target cells. The concentration of sera or antibodyrequired to neutralize 50% and 90% of input virus is indicated. MAb 48ddid not neutralize IIIB-BaL under any condition tested, and only 88%neutralization of 8x-BaL was achieved by this MAb at a concentration of200 ng/ml (as indicated by an *). MAb 50.1 is directed against BaLV3-loop as described by White-Scharf et al. (1993, Virology192:197-206).

[0299] The data disclosed herein demonstrate that HIV-1/IIIBx wasuniformly more sensitive to neutralization than the parental HIV-1/IIIB,in many cases by one-log or more (Table 2). HIV-1 -positive human sera,rabbit sera generated against IIIB gp120, and rabbit sera generatedagainst 8x gp120 all neutralized HIV-1/IIIBx far more efficiently thanHIV-1/IIIB. In addition, the data disclosed herein demonstrate thatvirions containing 8x-BaL Env were much more sensitive to neutralizationby the CD4i MAbs 17b and 48d than those containing IIIB-BaL Env, but8x-BaL Env virions did not demonstrate increased sensitivity toneutralization by an antibody recognizing the V3 loop of these viruses(Table 2). Thus, without wishing to be bound by theory, the datadisclosed herein demonstrate that increased exposure of the coreceptorbinding site, as well as increased exposure of CD4i epitopes, is likelyto account for the increased sensitivity of HIV-1/IIIBx toantibody-induced neutralization.

[0300] The prior art teaches that receptor binding triggersconformational changes in Env that activate its membrane fusionpotential. Binding to CD4 enables Env to interact with an appropriatecoreceptor (Wu et al., 1996, Nature 384:179-183; Trkola et al., 1996,Nature 384:184-187; Lapham et al., 1996, Science 274:602-605; Hill etal., 1997, J. Virol. 71:6296-6304), generally CCR5 or CXCR4, which isthought to result in additional conformational changes in Env thatultimately lead to membrane fusion and virus entry. Fusion is a criticalstep in virus infection, and understanding the structural intermediatesin Env that lead to this process may suggest the development of novelanti-viral strategies. Indeed, early clinical trials with a peptideinhibitor of the membrane fusion reaction have shown significantreductions in viral load (Kilby et al., 1998, Nature Med. 4:1302-1307).

[0301] The discovery of the viral coreceptors and the recently solvedcrystal structure of a gp120 core fragment have provided greaterunderstanding of the viral entry process and have identified newpotential targets for pharmacologic or immunologic intervention (Kwonget al., 1998, Nature 393:648-659; Rizzuto et al., 1998, Science280:1949-1953; Wyatt et al., 1998, Nature 393:705-710). In the case ofEnv, an exceptionally well conserved region in gp120 has been implicatedin CCR5 binding (Rizzuto et al., 1998, Science 280:1949-1953). Thisregion, located in the bridging sheet between the inner and outerdomains of gp120, lies between the base of the V3 loop and the V1/V2region (FIG. 12). Binding to CD4, which is known to reposition thesevariable regions (Wyatt et al., 1995, J. Virol. 69:5723-5733), may leadto exposure and/or formation of this highly conserved site. Neutralizingantibodies such as 17b bind to epitopes that overlap with this region(Rizzuto et al., 1998, Science 280:1949-1953; Wyatt et al., 1998, Nature393:705-710), thus serving as immunological surrogates for exposure ofthis domain and suggesting that, if properly presented, this region mayelicit broadly cross-reactive neutralizing antibodies. However, prior tothe present invention, there was no way to present this region to theimmune system in such a way to generate an immune response to thecoreceptor binding region of gp120.

[0302] A number of HIV-1, HIV-2, and SIV virus strains have beendescribed that bypass the normal viral entry process by interactingdirectly with CCR5 or CXCR4 to infect cells (Example 1; Edinger et al.,1997, Proc. Natl. Acad. Sci. U.S.A. 94:14742-14747; Endres et al., 1996,Cell 87:745-756). While CD4-independent viruses may impact viral tropismand pathogenesis, they also serve as useful tools for dissecting thevirus entry pathway. The data disclosed herein demonstrate two lines ofevidence indicating that the CD4-independent HIV-1 Env disclosed hereinexists in a stable, partially triggered state in which the conservedcoreceptor binding site is well exposed. First, 8x gp120 bound directlyto CXCR4 while the parental IIIB gp120 bound CXCR4 in a CD4-dependentmanner. Second, 8x gp120 bound much more rapidly to two CD4i MAbs thandid the parental CD4-dependent protein. However, as a consequence of afaster off-rate, the overall affinity of 8x gp120 for CD4i MAbs wassimilar to that of HIV-1 IIIB gp120, providing a striking example of howimportant differences in protein-protein interactions can be revealed bythe real time analysis afforded by the use of an optical biosensor. Thefaster off-rate exhibited by 8x relative to IIIB could be due to anumber of amino acid changes in the 8x protein in the vicinity of thecoreceptor binding site, including I423V, a residue which serves as acontact site for 17b (Kwong et al., 1998, Nature 393:648-659).

[0303] HIV-1 tropism is governed in large part by coreceptor choice. Theability of a virus to utilize CCRS, CXCR4, or both, largely dictates thetype of CD4-positive cells it can enter. The V3 loop in gp120 plays acritical role in coreceptor choice, with the V1/2 region playing a moresubsidiary role (Hoffman et al., 1998, Proc. Natl. Acad. Sci. U.S.A.95:11360-11365; Ross and Cullen, 1998, Proc. Natl. Acad. Sci. U.S.A.95:7682-7686; Speck et al., 1997, J. Virol. 71:7136-7139; Cocchi et al.,1996, Nature Med. 2:1244-1247; Cho et al., 1998, J. Virol. 72:2509-2515;Choe et al., 1996, Cell 85:1135-1148). The data disclosed hereindemonstrate that the determinants underlying coreceptor choice andCD4-independence in 8x Env are dissociable. Thus, 8x Env containing aV3-loop from an R5 Env maintains its CD4-independent phenotype but nowuses CCR5 rather than CXCR4 for cell-cell fusion and virus infection.Further, the data disclosed herein demonstrate that 8x-BaL gp120 is ableto bind to CCR5-expressing cells in the absence of CD4. These dataclearly demonstrate, for the first time, that coreceptor choice andCD4-independent use of a chemokine receptor are dissociable. These datasuggest, without wishing to be bound by theory, that the coreceptorbinding region can interact with both CXCR4 and CCR5, depending on thenature of the associated V3-loop (Rizzuto et al., 1998, Science280:1949-1953). Thus, in the context of the HIV-1 variant disclosedherein, the V3-loop can affect coreceptor choice in a partiallytriggered Env as well. The disclosure of the present invention willfacilitate the clarification of the respective roles the variableregions and the conserved binding site play in coreceptor interactionsand the identification of the domains in CCR5 and CXCR4 with which eachinteracts.

[0304] A particularly striking feature of the HIV-1/IIIBx swarm and the8x molecular clone was their sensitivity to neutralization byantibodies. HIV-1/IIIBx was approximately 10-fold more sensitive toneutralization by HIV-positive human sera as well as to rabbit seragenerated against either IIIB gp120 or 8x gp120. Without wishing to bebound by theory, the increased sensitivity of HIV-1/IIIBx to antibodymediated neutralization suggests that one or more neutralizationdeterminants in this partially triggered Env is more generallyaccessible to antibodies than in the parental, CD4-dependent Env. Theconserved coreceptor binding site is a likely target that may accountfor this phenotype, as indicated by the ability of CD4i MAbs to binddirectly to 8x Env and to efficiently neutralize 8x-BaL Env-pseudotypedvirions. In addition, as demonstrated by the data disclosed in Example 1herein, the 8x Env also exhibits a remarkable loss of 5 glycosylationsites relative to parental IIIB raising the possibility that the loss ofcarbohydrates could play a role in exposing this region. Despite theincreased neutralization of 8x-BaL by CD4i MAbs, increased exposure ofthe coreceptor binding site did not lead to increased sensitivity of8x-BaL to a MAb directed against the V3-loop (Table 2). Several otherneutralization sensitive viruses have been described recently, includinga SIVmac239 lacking glycosylation sites in the V1/V2 region, as well asa HIV-1 SF162 strain containing a deletion in V1 (Stamatatos andCheng-Mayer, 1998, J. Virol. 72:7840-7845). The present invention willfacilitate studies to determine whether the coreceptor binding sitewhich adjoins the V1/V2 stem is exposed in these viruses as well.

[0305] Numerous studies have shown that immunization with recombinantgp120 typically fails to generate broadly cross-reactive neutralizingantibodies, yet it is clear that such antibodies are generated in someindividuals as a consequence of virus infection (Burton and Motefiori,1997, AIDS 11 (Supp. A):S87-S98). Without wishing to be bound by theory,it may be that only strain-specific neutralization has been observedfollowing immunization by gp120 because conserved regions of Env aresequestered in CD4-dependent gp120s prior to CD4 binding. The datadisclosed herein suggest that if CD4-independence results from thepartially triggered form of gp120 which no longer requires the initialbinding to CD4 before the protein will bind to the chemokine receptorprotein, then exposure of the highly conserved chemokine receptorbinding site renders the virus more sensitive to neutralization. Moreimportantly, the data disclosed herein suggest indicate thatimmunization with Envs that are partially triggered thus stably exposingthe otherwise hidden chemokine receptor binding site may result in moreefficient generation of broadly cross-reactive neutralizing antibodiesdirected against this region. For this to occur, antibodies must begenerated that can access the coreceptor binding site in native,CD4-dependent Env proteins, perhaps after CD4-binding induces atriggered conformation in which access to this region is enhanced.Identifying determinants that render Envs CD4-independent and thatinfluence exposure of this region will make it possible tosystematically address the potential of this site to elicit neutralizingantibodies. It is important to note that while exposure of thecoreceptor binding site may be an important component of CD4-independentEnvs, other changes in Env are also likely to influence the ability toinfect cells in a CD4-independent manner (Example 1).

[0306] In addition, the ability to examine and map, at the molecularlevel, the chemokine receptor binding site determinant(s) willfacilitate the development of small-molecule inhibitors ofgp120/chemokine receptor binding.

[0307] A “small-molecule,” as the term is used herein, means a compound,whether synthetic or naturally occurring, including nucleic acids andpolypeptides, such as, but not limited to, peptidomimetics and ALX40-4C(Doranz et al., 1997, J. Exp. med. 186:1395-1400), which are capable ofinhibiting gp120 binding to a chemokine receptor protein, and which areless than about 1 kDa in size.

[0308] Therefore, the data disclosed herein have important implicationsin the development of effective antiviral therapeutics including, butnot limited to, antibody-based modalities. Thus, the present inventionshould be construed to encompass the development of a wide class ofcompounds as inhibitors of gp120/chemokine receptor proteininteractions.

[0309] Further, that the coreceptor binding site can be stably exposedmay have implications for viral entry. Without wishing to be bound bytheory, it is conceivable that exposure and/or formation of thecoreceptor binding site subsequent to CD4 binding could result in aconformation of Env that is relatively unstable, requiring interactionswith a coreceptor within a short period of time. For example, triggeringthe conformational change in the Semliki Forest virus spike glycoproteinby acid pH leads to rapid inactivation of the protein's membrane fusionpotential unless it can interact with its lipid coreceptors withinseveral minutes (Kielian, 1995, Advances in Virus Res. 45:113-151).However, the 8x Env protein, which exists in a partially triggered butstable state, suggests that exposure of the coreceptor binding site iscompatible with a long-lived triggered Env conformation. Thus,coreceptor binding could occur long after the conformational changesinduced by CD4 that make this event possible, perhaps accounting for theability of HIV-1 to infect cells that express very low levels ofcoreceptor when adequate levels of CD4 are present (Platt et al., 1998,J. Virol. 72:2855-2864; Kozak et al., 1997, J. Virol. 71:873-882). Thisdiscovery further emphasizes the potential of the present invention inthe development of antiviral therapeutics based on the inhibition ofHIV-1/chemokine receptor interactions since the triggered conformationmay potentially be present long enough for compounds blocking thenecessary determinants to effect the inhibition of virus binding to thehost cell receptors.

[0310] In conclusion, the data disclosed herein demonstrate that theCD4-independent phenotype of the 8x Env protein is associated withstable exposure of the coreceptor binding site. Thus, this proteinlikely represents a structural intermediate of the normal fusionprocess, and can be used to investigate the structural parameters thatinfluence the conformational changes that lead to membrane fusion.Importantly, the highly conserved nature of this stably exposed domainand the fact the neutralizing antibodies can be directed against itraise the possibility that this domain, if properly presented, can beused to elicit broadly cross-reactive neutralizing antibodies againstHIV-1.

[0311] The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

[0312] While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1 12 1 24 DNA Artificial Sequence Description of Artificial Sequence PCRprimer 1 cgcaacctat accaatagta gcaa 24 2 24 DNA Artificial SequenceDescription of Artificial Sequence PCR primer 2 cagtaagcca tccaatcacactac 24 3 726 PRT Human immunodeficiency virus type 1 3 Met Arg Val LysGlu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5 10 15 Trp Gly ThrMet Leu Leu Gly Met Leu Met Ile Cys Asn Ala Thr Glu 20 25 30 Lys Leu TrpVal Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 35 40 45 Thr Thr ThrLeu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Thr Glu 50 55 60 Val His AsnVal Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 75 80 Pro GlnGlu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp 85 90 95 Lys AsnAsp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp 100 105 110 AspGln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ser 115 120 125Leu Lys Cys Thr Asp Leu Lys Asn Asp Thr Asn Thr Asn Ser Gly Ser 130 135140 Gly Arg Met Ile Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn 145150 155 160 Ile Ser Thr Ser Lys Arg Ser Lys Val Lys Lys Glu Tyr Ala PhePhe 165 170 175 Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp Pro Thr SerTyr Thr 180 185 190 Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala CysPro Lys Val 195 200 205 Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala ProAla Gly Phe Ala 210 215 220 Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn GlyThr Gly Pro Cys Thr 225 230 235 240 Asn Val Ser Thr Val Gln Cys Thr HisGly Ile Arg Pro Val Val Ser 245 250 255 Thr Gln Leu Leu Leu Asn Gly SerLeu Ala Glu Glu Glu Val Val Ile 260 265 270 Arg Ser Val Asn Phe Thr AspAsn Ala Lys Thr Ile Ile Val Gln Leu 275 280 285 Asn Thr Ser Val Glu IleAsn Cys Thr Lys Pro Asn Asn Asn Thr Arg 290 295 300 Lys Arg Ile Arg IleHis Arg Gly Pro Gly Arg Ala Phe Val Thr Val 305 310 315 320 Gly Lys IleGly Asn Met Arg Gln Ala His Cys Asn Ile Ser Arg Ala 325 330 335 Lys TrpSer Asn Thr Leu Lys Gln Ile Ala Ser Lys Leu Arg Glu Gln 340 345 350 PheGly Asn Asn Lys Thr Ile Ile Phe Lys Gln Ser Ser Gly Gly Asp 355 360 365Pro Glu Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr 370 375380 Cys Lys Ser Thr Gln Leu Phe Asn Ser Thr Trp Ser Thr Lys Gly Ser 385390 395 400 Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu Pro Cys Arg IleLys 405 410 415 Gln Val Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met TyrAla Pro 420 425 430 Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser Asn Ile ThrGly Leu Leu 435 440 445 Leu Thr Arg Asp Gly Gly Asn Ser Asn Asn Glu SerGlu Ile Phe Arg 450 455 460 Pro Gly Gly Gly Asp Met Arg Asp Asn Trp ArgSer Glu Leu Tyr Lys 465 470 475 480 Tyr Lys Val Val Lys Ile Glu Pro LeuGly Val Ala Pro Thr Lys Ala 485 490 495 Lys Arg Arg Val Val Gln Arg GluLys Arg Ala Val Gly Ile Gly Ala 500 505 510 Leu Phe Leu Gly Phe Leu GlyAla Ala Gly Ser Thr Met Gly Ala Ala 515 520 525 Ser Met Ala Leu Thr ValGln Ala Arg Gln Ser Leu Ser Gly Ile Val 530 535 540 Gln Gln Gln Asn AsnLeu Leu Arg Ala Ile Glu Ala Gln Gln His Leu 545 550 555 560 Leu Gln LeuThr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Ile Leu 565 570 575 Ala ValGlu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly 580 585 590 CysSer Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser 595 600 605Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn Asn Met Thr Trp Met 610 615620 Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His Ser Leu 625630 635 640 Ile Glu Glu Ser Gln Ile Gln Gln Glu Met Asn Glu Gln Glu LeuLeu 645 650 655 Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asn IleThr Asn 660 665 670 Trp Leu Trp Tyr Ile Lys Leu Phe Ile Met Ile Val GlyGly Leu Val 675 680 685 Gly Leu Arg Ile Val Phe Ala Val Leu Ser Val ValLys Lys Leu Gly 690 695 700 Arg Asp Ile His His Tyr Arg Phe Arg Pro ThrSer Gln His Arg Gly 705 710 715 720 Asp Thr Gly Pro Lys Glu 725 4 2184DNA Human immunodeficiency virus type 1 4 atgagagtga aggagaaatatcagcacttg tggagatggg ggtggagatg gggcaccatg 60 ctccttggga tgttgatgatctgtaatgct acagaaaaat tgtgggtcac agtctattat 120 ggggtacctg tgtggaaggaagcaaccacc actctatttt gtgcatcaga tgctaaagca 180 tatgaaacag aggtacataatgtttgggcc acacatgcct gtgtacccac agaccccaac 240 ccacaagaag tagtattggtaaatgtgaca gaaaatttta acatgtggaa aaatgacatg 300 gtagaacaga tgcatgaggatataatcagt ttatgggatc aaagcctaaa gccatgtgta 360 aaattaaccc cactctgtgttagtttaaag tgcactgatt tgaagaatga tactaatacc 420 aatagtggta gcgggagaatgataatggag aaaggagaga taaaaaactg ctctttcaat 480 atcagcacaa gcaaaagaagtaaggtgaag aaagaatatg cattttttta taaacttgat 540 ataataccaa tagataatgatcctaccagc tatacgttga caagttgtaa cacctcagtc 600 attacacagg cctgtccaaaggtatccttt gagccaattc ccatacatta ttgtgccccg 660 gctggttttg cgattctaaaatgtaataat aagacgttca atggaacagg accatgtaca 720 aatgtcagca cagtacaatgtacacatgga attaggccag tagtatcaac tcaactgctg 780 ttaaatggca gtctagcagaagaagaggta gtaattagat ctgtcaattt cacggacaat 840 gctaaaacca taatagtacagctgaacaca tctgtagaaa ttaattgtac aaaacccaac 900 aacaatacaa gaaaaagaatccgtatccat agaggaccag ggagagcatt tgttacagta 960 ggaaaaatag gaaatatgagacaagcacat tgtaacatta gtagagcaaa atggagtaac 1020 actttaaaac agatagctagcaaattaaga gaacaatttg gaaataataa aacaataatc 1080 tttaagcagt cctcaggaggggacccagaa attgtaacgc acagttttaa ttgtggaggg 1140 gaatttttct actgtaagtcaacacaactg tttaatagta cttggagtac taaagggtca 1200 aataacactg aaggaagtgacacaatcacc ctcccatgca gaataaaaca agttataaac 1260 atgtggcagg aagtaggaaaagcaatgtat gcccctccca tcagtggaca aattagatgt 1320 tcatcaaata ttacagggctgctattaaca agagatggtg gtaatagcaa caatgagtcc 1380 gagatcttca gacctggaggaggagatatg agggacaatt ggagaagtga attatataaa 1440 tataaagtag taaaaattgaaccattagga gtagcaccca ccaaggcaaa gagaagagtg 1500 gtgcagagag aaaaaagagcagtgggaata ggagctttgt tccttgggtt cttgggagca 1560 gcaggaagca ctatgggcgcagcgtcaatg gcgctgacgg tacaggccag acaatcattg 1620 tctggtatag tgcagcagcagaacaatctg ctgagggcta ttgaggcgca acagcatctg 1680 ttgcaactca cagtctggggcatcaagcag ctccaggcaa gaatcctggc tgtggaaaga 1740 tacctaaagg atcaacagctcctggggatt tggggttgct ctggaaaact catttgcacc 1800 actgctgtgc cttggaatgctagttggagt aataaatctc tggaacagat ttggaataac 1860 atgacctgga tggagtgggacagagaaatt aacaattaca caagcttaat acactcctta 1920 attgaagaat cgcaaatccagcaagaaatg aatgaacaag aattattgga attagataaa 1980 tgggcaagtt tgtggaattggtttaacata acaaattggc tgtggtatat aaaattattc 2040 ataatgatag taggaggcttggtaggttta agaatagttt ttgctgtact ttctgtagtg 2100 aaaaagttag gcagggatattcaccattat cgtttcagac ccacctccca acaccgaggg 2160 gacccgacag gcccgaaggaatag 2184 5 30 DNA Artificial Sequence Description of ArtificialSequencePCR primer 5 cctcaggagg ggacccagaa attgtaacgc 30 6 30 DNAArtificial Sequence Description of Artificial SequencePCR primer 6gcgttacaat ttctgggtcc cctcctgagg 30 7 31 DNA Artificial SequenceDescription of Artificial SequencePCR primer 7 ggcaggaagt agaaaaagcaatgtatgccc c 31 8 31 DNA Artificial Sequence Description of ArtificialSequencePCR primer 8 ggggcataca ttgctttttc tacttcctgc c 31 9 31 DNAArtificial Sequence Description of Artificial SequencePCR primer 9ggcaggaagt aggaaaagca atgtatgccc c 31 10 31 DNA Artificial SequenceDescription of Artificial SequencePCR primer 10 ggggcataca ttgcttttcctacttcctgc c 31 11 856 PRT Human immunodeficiency virus type 1 11 MetArg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg 1 5 10 15Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Asn Ala Thr Glu 20 25 30Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 35 40 45Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 7580 Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asp Met Trp 85 9095 Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp 100105 110 Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ser115 120 125 Leu Lys Cys Thr Asp Leu Lys Asn Asp Thr Asn Thr Asn Ser SerSer 130 135 140 Gly Arg Met Ile Met Glu Lys Gly Glu Ile Lys Asn Cys SerPhe Asn 145 150 155 160 Ile Ser Thr Ser Ile Arg Gly Lys Val Gln Lys GluTyr Ala Phe Phe 165 170 175 Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn AspThr Thr Ser Tyr Ser 180 185 190 Leu Thr Ser Cys Asn Thr Ser Val Ile ThrGln Ala Cys Pro Lys Val 195 200 205 Ser Phe Glu Pro Ile Pro Ile His TyrCys Ala Pro Ala Gly Phe Ala 210 215 220 Ile Leu Lys Cys Asn Asn Lys ThrPhe Asn Gly Thr Gly Pro Cys Thr 225 230 235 240 Asn Val Ser Thr Val GlnCys Thr His Gly Ile Arg Pro Val Val Ser 245 250 255 Thr Gln Leu Leu LeuAsn Gly Ser Leu Ala Glu Glu Glu Val Val Ile 260 265 270 Arg Ser Val AsnPhe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu 275 280 285 Asn Thr SerVal Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg 290 295 300 Lys ArgIle Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile 305 310 315 320Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys Asn Ile Ser Arg Ala 325 330335 Lys Trp Asn Asn Thr Leu Lys Gln Ile Asp Ser Lys Leu Arg Glu Gln 340345 350 Phe Gly Asn Asn Lys Thr Ile Ile Phe Lys Gln Ser Ser Gly Gly Asp355 360 365 Pro Glu Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe PheTyr 370 375 380 Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe Asn SerThr Trp 385 390 395 400 Ser Thr Glu Gly Ser Asn Asn Thr Glu Gly Ser AspThr Ile Thr Leu 405 410 415 Pro Cys Arg Ile Lys Gln Ile Ile Asn Met TrpGln Lys Val Gly Lys 420 425 430 Ala Met Tyr Ala Pro Pro Ile Ser Gly GlnIle Arg Cys Ser Ser Asn 435 440 445 Ile Thr Gly Leu Leu Leu Thr Arg AspGly Gly Asn Ser Asn Asn Glu 450 455 460 Ser Glu Ile Phe Arg Pro Gly GlyGly Asp Met Arg Asp Asn Trp Arg 465 470 475 480 Ser Glu Leu Tyr Lys TyrLys Val Val Lys Ile Glu Pro Leu Gly Val 485 490 495 Ala Pro Thr Lys AlaLys Arg Arg Val Val Gln Arg Glu Lys Arg Ala 500 505 510 Val Gly Ile GlyAla Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser 515 520 525 Thr Met GlyAla Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu 530 535 540 Leu SerGly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu 545 550 555 560Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu 565 570575 Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu 580585 590 Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val595 600 605 Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile TrpAsn 610 615 620 His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn TyrThr Ser 625 630 635 640 Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn GlnGln Glu Lys Asn 645 650 655 Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp AlaSer Leu Trp Asn Trp 660 665 670 Phe Asn Ile Thr Asn Trp Leu Trp Tyr IleLys Leu Phe Ile Met Ile 675 680 685 Val Gly Gly Leu Val Gly Leu Arg IleVal Phe Ala Val Leu Ser Ile 690 695 700 Val Asn Arg Val Arg Gln Gly TyrSer Pro Leu Ser Phe Gln Thr His 705 710 715 720 Leu Pro Thr Pro Arg GlyPro Asp Arg Pro Glu Gly Ile Glu Glu Glu 725 730 735 Gly Gly Glu Arg AspArg Asp Arg Ser Ile Arg Leu Val Asn Gly Ser 740 745 750 Leu Ala Leu IleTrp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr 755 760 765 His Arg LeuArg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu Leu 770 775 780 Leu GlyArg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu Leu 785 790 795 800Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn 805 810815 Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val 820825 830 Val Gln Gly Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg835 840 845 Gln Gly Leu Glu Arg Ile Leu Leu 850 855 12 759 PRT Humanimmunodeficiency virus type 1 12 Met Arg Val Lys Glu Lys Tyr Gln His LeuTrp Arg Trp Gly Trp Arg 1 5 10 15 Trp Gly Thr Met Leu Leu Gly Met LeuMet Ile Cys Asn Ala Thr Glu 20 25 30 Lys Leu Trp Val Thr Val Tyr Tyr GlyVal Pro Val Trp Lys Glu Ala 35 40 45 Thr Thr Thr Leu Phe Cys Ala Ser AspAla Lys Ala Tyr Glu Thr Glu 50 55 60 Val His Asn Val Trp Ala Thr His AlaCys Val Pro Thr Asp Pro Asn 65 70 75 80 Pro Gln Glu Val Val Leu Val AsnVal Thr Glu Asn Phe Asn Met Trp 85 90 95 Lys Asn Asp Met Val Glu Gln MetHis Glu Asp Ile Ile Ser Leu Trp 100 105 110 Asp Gln Ser Leu Lys Pro CysVal Lys Leu Thr Pro Leu Cys Val Ser 115 120 125 Leu Lys Cys Thr Asp LeuLys Asn Asp Thr Asn Thr Asn Ser Ser Ser 130 135 140 Gly Arg Met Ile MetGlu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn 145 150 155 160 Ile Ser ThrSer Lys Arg Gly Lys Val Lys Lys Glu Tyr Ala Phe Phe 165 170 175 Tyr LysLeu Asp Ile Ile Pro Ile Asp Asn Asp Pro Thr Ser Tyr Thr 180 185 190 LeuThr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val 195 200 205Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala 210 215220 Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro Cys Thr 225230 235 240 Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val ValSer 245 250 255 Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu ValVal Ile 260 265 270 Arg Ser Val Asn Phe Thr Asp Asn Ala Lys Thr Ile IleVal Gln Leu 275 280 285 Asn Thr Ser Val Glu Ile Asn Cys Thr Lys Pro AsnAsn Asn Thr Arg 290 295 300 Lys Arg Ile Arg Ile Gln Arg Gly Pro Gly ArgAla Phe Val Thr Val 305 310 315 320 Gly Lys Ile Gly Asn Met Arg Gln AlaHis Cys Asn Ile Ser Arg Ala 325 330 335 Lys Trp Ser Asn Thr Leu Lys GlnIle Ala Ser Lys Leu Arg Glu Gln 340 345 350 Phe Gly Asn Asn Lys Thr IleIle Phe Lys Gln Ser Ser Gly Gly Asp 355 360 365 Pro Glu Ile Val Thr HisSer Phe Asn Cys Gly Gly Glu Phe Phe Tyr 370 375 380 Cys Lys Ser Thr GlnLeu Phe Asn Ser Thr Trp Ser Thr Lys Gly Ser 385 390 395 400 Asn Asn ThrGlu Gly Ser Asp Thr Ile Thr Leu Pro Cys Arg Ile Lys 405 410 415 Gln IleIle Asn Met Trp Gln Lys Val Glu Lys Ala Met Tyr Ala Pro 420 425 430 ProIle Ser Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu 435 440 445Leu Thr Arg Asp Gly Gly Asn Asn Asn Asn Glu Ser Glu Ile Phe Arg 450 455460 Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys 465470 475 480 Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr LysAla 485 490 495 Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala Val Gly IleGly Ala 500 505 510 Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr MetGly Ala Ala 515 520 525 Ser Met Ala Leu Thr Val Gln Ala Arg Gln Ser LeuSer Gly Ile Val 530 535 540 Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile GluAla Gln Gln His Leu 545 550 555 560 Leu Gln Leu Thr Val Trp Gly Ile LysGln Leu Gln Ala Arg Ile Leu 565 570 575 Ala Val Glu Arg Tyr Leu Lys AspGln Gln Leu Leu Gly Ile Trp Gly 580 585 590 Cys Ser Gly Lys Leu Ile CysThr Thr Ala Val Pro Trp Ser Ala Ser 595 600 605 Trp Ser Asn Lys Ser LeuGlu Gln Ile Trp Asn Asn Met Thr Trp Met 610 615 620 Glu Trp Asp Arg GluIle Asn Asn Tyr Thr Ser Leu Ile His Ser Leu 625 630 635 640 Ile Glu GluSer Gln Asn Gln Gln Glu Met Asn Glu Gln Glu Leu Leu 645 650 655 Glu LeuAsp Lys Trp Ala Ser Leu Trp Asn Trp Phe Ile Ile Ser Ser 660 665 670 TrpLeu Trp Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Val 675 680 685Gly Leu Arg Ile Val Phe Ala Val Phe Ser Ile Val Asn Arg Val Arg 690 695700 Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr His Leu Pro Ile Pro Lys 705710 715 720 Gly Pro Asp Arg Pro Lys Arg Ile Leu Asn Thr Tyr Leu Gly ArgSer 725 730 735 Ala Glu Pro Val Pro Leu Gln Leu Pro Pro Leu Glu Arg LeuSer Gly 740 745 750 Thr Leu Asp Cys Asn Lys Asp 755

What is claimed is:
 1. An isolated nucleic acid encoding aCD4-independent human immunodeficiency virus-1 (HIV-1) env, or a mutant,derivative, or fragment thereof.
 2. The isolated nucleic acid of claim1, wherein said nucleic acid shares at least about 98% homology with thenucleic acid having the nucleotide sequence of SEQ ID NO:4.
 3. Theisolated nucleic acid of claim 2, wherein said nucleic acid is selectedfrom the group consisting of an HIV-1/IIIBx env, and an HIV-1/IIIBx 8x(8x) env.
 4. The isolated nucleic acid of claim 3, wherein said nucleicacid is an HIV-1/IIIBx 8x env.
 5. An isolated nucleic acid encoding aCD4-independent HIV env having the nucleotide sequence of SEQ ID NO:4.6. An isolated nucleic acid comprising a portion of a HIV-1 env genewhich confers CD4 independence on at least one HIV-1 env clone.
 7. Achimeric nucleic acid comprising a first portion and a second portion,said first portion encoding at least a portion of an HIV-1/IIIBx 8x envcoding sequence and said second portion encoding at least a portion ofan HIV-1 env coding sequence which is not an 8x env.
 8. The chimericnucleic acid of claim 7, wherein said second portion is an env codingsequence selected from the group consisting of an S10 env, an HXB2 env,a BaL env, and an IIIB env.
 9. The chimeric nucleic acid of claim 7,wherein said second portion comprises a chemokine receptor binding siteselected from the group consisting of a CXCR4 chemokine receptor bindingsite, and a CCR5 chemokine receptor binding site.
 10. The chimericnucleic acid of claim 9, wherein said second portion comprises a V3-loopcoding sequence selected from the group consisting of a V3-loop for aCXCR4 chemokine receptor binding site, and a V3-loop for a CCR5chemokine receptor binding site.
 11. An isolated HIV-1 gp120 polypeptidecomprising a stably exposed chemokine coreceptor binding site.
 12. Anisolated polypeptide comprising an HIV-1/IIIBx 8x Env.
 13. The isolatedpolypeptide of claim 12, wherein said polypeptide shares at least about98% homology with SEQ ID NO:3.
 14. The isolated polypeptide of claim 13comprising the amino acid sequence of SEQ ID NO:3.
 15. A chimeric HIV-1Env polypeptide comprising a gp120 polypeptide wherein said chimericpolypeptide comprises a first portion comprising an HIV-1/IIIBx 8xgp120, said chimeric polypeptide further comprising a second portioncomprising a gp120 from an HIV-1 other than HIV-1/IIIBx 8x.
 16. Achimeric HIV-1 Env polypeptide wherein said polypeptide isCD4-independent, and further wherein said polypeptide comprises achemokine receptor binding site selected from the group consisting of aCXCR4 chemokine receptor binding site, and a CCR5 chemokine receptorbinding site.
 17. The chimeric polypeptide of claim 16, wherein saidsecond portion comprises a V3-loop selected from the group consisting ofa HXB V3-loop, an 8x V3-loop, a BaL V3-loop, a YU-2 V3-loop, and an 89.6V3-loop.
 18. A composition comprising a CD4-independent HIV-1 Envcomprising a gp120 polypeptide comprising a stably exposed chemokinereceptor binding site wherein said HIV-1 is more sensitive to antibodyneutralization than an otherwise identical HIV-1 which does not comprisea stably exposed chemokine receptor binding site.
 19. A pharmaceuticalcomposition comprising a CD4-independent HIV-1 Env protein, wherein saidHIV-1 Env comprises at least one mutation causing the chemokinecoreceptor binding site to be stably exposed.
 20. The composition ofclaim 21, wherein said HIV-1 Env is HIV-1/IIIBx 8x.
 21. A vaccinecomprising an immunogenic dose of a CD4-independent HIV-1 Env.
 22. Thevaccine of claim 21, wherein said HIV-1 Env is selected from the groupconsisting of a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1Env, and a cell expressing HIV-1 Env.
 23. A vector comprising theisolated nucleic acid of claim
 1. 24. A vector comprising the isolatednucleic acid of claim
 6. 25. A vector comprising the isolated nucleicacid of claim
 7. 26. A cell comprising the isolated nucleic acid ofclaim
 1. 27. A cell comprising the isolated nucleic acid of claim
 6. 28.A cell comprising the isolated nucleic acid of claim
 7. 29. A cellcomprising the isolated polypeptide of claim
 11. 30. A cell comprisingthe isolated polypeptide of claim
 12. 31. A cell comprising the isolatedpolypeptide of claim
 15. 32. A cell comprising the isolated polypeptideof claim
 16. 33. A cell comprising the isolated polypeptide of claim 17.34. A cell comprising the composition of claim
 18. 35. A method ofidentifying an amino acid residue of an HIV-1 Env protein which isinvolved in CD4 independence, said method comprising obtaining afull-length env coding sequence from an Env clone which isCD4-independent and replacing at least a portion of the said env codingsequence with a coding sequence from an Env clone which is CD4-dependentto form a chimera, wherein when said chimera is CD4-dependent it is anindication that said portion of said env coding sequence is involved inCD4-independence, thereby identifying an amino acid residue involved inCD4-independence.
 36. A method of eliciting an immune response to aHIV-1 chemokine receptor binding site in a mammal, said methodcomprising administering an immunogenic dose of a CD4-independent HIV-1Env protein to a mammal, wherein said protein comprises a stably exposedchemokine receptor binding site, thereby eliciting an immune response toa HIV-1 chemokine receptor binding site in said mammal.
 37. A method ofidentifying a compound which affects exposure of an HIV-1 gp120chemokine receptor binding site, said method comprising contacting acell with said compound prior to or contemporaneous with contacting saidcell with a labeled gp120 with or without pre-incubation of said gp120with soluble CD4, measuring the amount of label bound to said cell, andcomparing the amount of label bound to said cell contacted with saidcompound to the amount of label bound to an otherwise identical cell notcontacted with said compound, wherein a higher or lower amount of labelbound to said cell contacted with said compound compared with the amountof label bound to said otherwise identical cell not contacted with saidcompound, is an indication that said compound affects exposure of anHIV-1 gp120 chemokine receptor binding site.
 38. A method of identifyinga small-molecule which inhibits binding of an HIV-1 gp120, using itschemokine receptor binding site, to a chemokine receptor, said methodcomprising contacting a cell with said molecule prior to orcontemporaneous with contacting said cell with labeled gp120 with orwithout pre-incubation of said gp120 with soluble CD4, measuring theamount of label bound to said cell, and comparing the amount of labelbound to said cell contacted with said molecule with the amount of labelbound to an otherwise identical cell not contacted with said molecule,wherein a lower amount of label bound to said cell contacted with saidmolecule compared with the amount of label bound to said otherwiseidentical cell not contacted with said molecule, is an indication thatsaid molecule inhibits binding of an HIV-1 gp120 using its chemokinereceptor binding site to a chemokine receptor.
 39. A method of producinga CD4-independent chimeric HIV-1 Env clone comprising a variablechemokine receptor binding site, said method comprising replacing thehypervariable V3-loop of the CD4-independent Env clone with the V3 loopof another HIV-1, wherein said V3-loop of another HIV-1 comprises adifferent chemokine receptor binding site than that of saidCD4-independent Env clone.
 40. The method of claim 39, wherein saidCD4-independent clone is selected from the group consisting ofHIV-1/IIIBx, and HIV-1/IIIBx 8x.
 41. The method of claim 40, whereinsaid V3-loop from another HIV-1 is selected from the group consisting ofa V3-loop from HIV-1/BaL, a V3-loop from HIV-1/YU-2, a V3-loop fromHIV-1/ADA, and a V3-loop from HIV-1/89.6.
 42. A method of inhibitingHIV-1 gp120 binding, using its chemokine receptor binding site, to achemokine receptor, said method comprising contacting said gp120 with asmall-molecule identified using the method of claim 37, therebyinhibiting HIV-1 gp120 binding, using its chemokine receptor bindingsite, to a chemokine receptor.
 43. A method of inhibiting HIV-1infection of a cell, said method comprising contacting said cell with asmall-molecule which inhibits binding of an HIV-1 gp120 using itschemokine receptor binding site to a chemokine receptor, wherein saidsmall-molecule is identified using the method of claim 38, therebyinhibiting HIV-1 infection of a cell.
 44. A composition comprising aCD4-independent HIV-1 Env and at least one compound used to treat HIVinfection in a pharmaceutically suitable carrier.
 45. The composition ofclaim 44, wherein said HIV-1 Env is selected from the group consistingof a HIV-1 Env polypeptide, a nucleic acid encoding HIV-1 Env, and acell expressing HIV-1 env.
 46. The composition of claim 44, wherein saidcompound used to treat HIV infection is selected from the groupconsisting of a protease inhibitor, a reverse transcriptase nucleosideanalog inhibitor, a reverse transcriptase non-nucleoside analoginhibitor, an interferon, AZT, interleukin-2, and a cytokine.
 47. Amethod of treating HIV-1 infection in a human, said method comprisingadministering an immunogenic dose of a CD4-independent HIV-1 Env to anHIV-1 infected human, thereby treating HIV-1 infection in said human.48. The method of claim 47, wherein said HIV-1 Env is selected from thegroup consisting of a HIV-1 Env polypeptide, a nucleic acid encodingHIV-1 Env, and a cell expressing HIV-1 env.
 49. The method of claim 48,said method further comprising administering a compound used to treatHIV infection.
 50. The method of claim 49, wherein said compound used totreat HIV infection is selected from the group consisting of a proteaseinhibitor, a reverse transcriptase nucleoside analog inhibitor, areverse transcriptase non-nucleoside analog inhibitor, an interferon,AZT, interleukin-2, and a cytokine.
 51. The method of claim 50, whereinsaid compound is administered to said human before, during or afteradministration of said CD4-independent HIV-1 Env.